Polymeric prodrug with a self-immolative linker

ABSTRACT

A cascade carrier linked prodrug is described which comprises a biologically active moiety and a masking group having at least one nucleophile and being distinct from the carrier.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application, under 35 U.S.C. §371,of PCT/EP2005/003061 filed Mar. 22, 2005, which claims benefit under 35U.S.C. §119 to European Patent Application No. 04075892.2, filed on Mar.23, 2004, British Patent Application No. 0415043.9, filed on Jul. 5,2004, and European Patent Application No. 04019293.2, filed on Aug. 13,2004.

BACKGROUND OF THE INVENTION

Typically, polymers are either used in a non-covalent fashion, with thedrug compound physicochemically formulated into a solvent-polymermixture, or by permanent covalent attachment of a polymer reagent to oneof the drug's functional groups.

Non-covalent drug encapsulation has been applied to depot formulationsfor long-acting release profiles. Typically, drug is mixed with polymermaterial and processed in such fashion, that the drug becomesdistributed throughout the bulk polymer material. Such polymer-proteinaggregates may be shaped as microparticles which are administered as aninjectable suspension or they are formulated as gels which areadministered in a single bolus injection. Drug release occurs when thepolymer swells or degradation of the polymer allows for diffusion of thedrug to the exterior. Such degradation processes may be autohydrolyticor enzyme-catalyzed. An example for a marketed drug based on bolusadministration of a drug-polymer gel is Lupron Depot. An example for amarketed drug based on suspended microparticles is Nutropin Depot.

A disadvantage of the non-covalent approach is that in order to preventuncontrolled, burst-type release of the drug, encapsulation has to behighly efficient by creating a sterically highly crowded environment.Restraining the diffusion of an unbound, water soluble drug moleculerequires strong van der Waals contacts, frequently mediated throughhydrophobic moieties. Many conformationally sensitive therapeutics suchas proteins or peptides are rendered dysfunctional during theencapsulation process and/or during subsequent storage. In addition,such amino-containing drug compounds readily undergo side reactions withpolymer degradation products (D. H. Lee et al., J. Contr. Rel., 2003,92, 291-299). Furthermore, dependence of the release mechanism uponbiodegradation may cause interpatient variability.

Alternatively, drugs may be conjugated to polymers through permanentcovalent bonds. This approach is applied to various classes ofmolecules, from so-called small molecules, through natural products upto larger proteins.

Many small molecule medicinal agents, like alkaloids and anti-tumoragents, show low solubility in aqueous fluids. One way to solubilizethese small molecule compounds is to conjugate them to hydrophilicpolymers. A variety of water-soluble polymers, such as human serumalbumin, dextran, lectins, poly(ethylene glycol) (PEG),poly(styrene-co-maleic anhydride), poly(N-hydroxypropylmethacrylamide),poly(divinyl ether-co-maleic anhydride), hyaluronic acid have beendescribed for this purpose (R. Duncan, Nature Rev. Drug Disc., 2003, 2,347-360).

A major challenge in cancer therapy is to selectively target cytotoxicagents to tumor cells. A promising method to accumulate small moleculeanticancer agents in tumor tissue and decrease undesirable side effectsof these agents is the attachment of the cytotoxin to a macromolecularcarrier. The passive targeting of polymeric drug conjugates to tumors isbased on the so-called enhanced permeability and retention effect (EPR)as described by Matsumura, Y. and Maeda, H., in Cancer Res., 1986, vol6, pp 6387-6392. As a result, several polymer-drug conjugates haveentered clinical trial as anticancer agents.

Covalent modification of biological molecules with poly(ethylene glycol)has been extensively studied since the late 1970s. So-called PEGylatedproteins have shown improved therapeutic efficacy by increasingsolubility, reducing immunogenicity, and increasing circulationhalf-live in vivo due to reduced renal clearance and proteolysis byenzymes (see, for example, Caliceti P., Veronese F. M., Adv. Drug Deliv.Rev. 2003, 55, 1261-1277).

However, many medicinal agents such as INFalfa2, saquinavir orsomatostatin are inactive or show decreased biological activity when apolymer is covalently conjugated to the drug molecule (T. Peleg-Shulmanet al., J. Med. Chem., 2004, 47, 4897-4904).

In order to avoid shortcomings imposed by either non-covalent polymermixtures or permanent covalent attachment, it may be preferable toemploy a prodrug approach for chemical conjugation of drug to polymercarrier. In such polymeric prodrugs, the biologically active moietiesare typically linked to the polymeric carrier moiety by a temporary bondformed between the carrier moiety and a hydroxy, amino or carboxy groupof the drug molecule (such as is shown in FIG. 1).

Prodrugs are therapeutic agents that are almost inactive per se but arepredictably transformed into active metabolites (see B. Testa, J. M:Mayer in Hydrolysis in Drug and Prodrug Metabolism, Wiley-VCH, 2003,page 4). The carrier prodrug approach may be applied in such a fashionthat the medicinal agent is released in vivo from the polymer in orderto regain its biological activity. The reduced biological activity ofthe prodrug as compared to the released drug is of advantage if a slowor controlled release of the drug is desired. In this case, a relativelylarge amount of prodrug may be administered without concomitant sideeffects and the risk of overdosing. Release of the drug occurs overtime, thereby reducing the necessity of repeated and frequentadministration of the drug.

Prodrug activation may occur by enzymatic or non-enzymatic cleavage ofthe temporary bond between the carrier and the drug molecule, or asequential combination of both, i.e. an enzymatic step followed by anon-enzymatic rearrangement. In an enzyme-free in vitro environment suchas an aqueous buffer solution, a temporary bond such as an ester oramide may undergo hydrolysis, but the corresponding rate of hydrolysismay be much too slow and not therapeutically useful. In an in vivoenvironment, esterases or amidases are typically present and may causesignificant catalytic acceleration of the kinetics of hydrolysis fromtwofold up to several orders of magnitude (see, for example, R. B.Greenwald et al. J. Med. Chem. 1999, 42 (18), 3857-3867).

Definitions Based on IUPAC (as given underhttp://www.chem.qmul.ac.uk/iupac/medchem/ (accessed on 8 Mar. 2004)

Prodrug

A prodrug is any compound that undergoes biotransformation beforeexhibiting its pharmacological effects. Prodrugs can thus be viewed asdrugs containing specialized non-toxic protective groups used in atransient manner to alter or to eliminate undesirable properties in theparent molecule.

Carrier-Linked Prodrug (Carrier Prodrug)

A carrier-linked prodrug is a prodrug that contains a temporary linkageof a given active substance with a transient carrier group that producesimproved physicochemical or pharmacokinetic properties and that can beeasily removed in vivo, usually by a hydrolytic cleavage. This is showngraphically in FIG. 1.

Cascade Prodrug

A cascade prodrug is a carrier prodrug for which the cleavage of thecarrier group becomes effective only after unmasking an activatinggroup.

Polymeric Cascade Prodrug

A polymeric cascade prodrug is a carrier prodrug that contains atemporary linkage of a given active substance with a transient polymericcarrier group for which the cleavage of the carrier becomes effectiveonly after unmasking an activating group.

Bioprecursor Prodrug

A bioprecursor prodrug is a prodrug that does not imply the linkage to acarrier group, but results from a molecular modification of the activeprinciple itself. This modification generates a new compound, able to betransformed metabolically or chemically, the resulting compound beingthe active principle.

Biotransformation

Biotransformation is the chemical conversion of substances by livingorganisms or enzyme preparations.

Prodrugs fall in two classes, bioprecursors and carrier-linked prodrugs.Bioprecursors do not contain a carrier group and are activated by themetabolic creation of a functional group. In carrier-linked prodrugs theactive substance is linked to a carrier moiety by a temporary linkage.This invention is concerned with polymeric carrier-linked ormacromolecular prodrugs, where the carrier itself is a macromoleculesuch as a carrier protein or polysaccharide or polyethylene glycol.Specifically, the invention relates to polymeric carrier-linked prodrugsfor which this cleavage between polymer and drug proceeds in two stepsaccording to a cascade mechanism.

Cleavage of a carrier prodrug generates a molecular entity (drug) ofincreased bioactivity and at least one side product, the carrier. Thisside product may be biologically inert (for instance PEG) or may havetargeting properties (for instance antibodies). After cleavage, thebioactive entity will reveal at least one previously conjugated andthereby protected functional group, and the presence of this grouptypically contributes to the drug's bioactivity.

In order to implement a prodrug strategy, at least one certainfunctional group in the drug molecule is employed for attachment of thecarrier polymer. Preferred functional groups are hydroxyl or aminogroups. Consequently, both the attachment chemistry and hydrolysisconditions vary greatly between these two functionalities.

In a simple one-step mechanism, the prodrug's temporary linkage ischaracterized by an intrinsic liability or enzyme dependence. Thesusceptibility of this linkage to hydrolysis in an aqueous environmentwith our without enzyme catalysis controls the cleavage kinetics betweenpolymeric carrier and drug. Numerous macromolecular prodrugs aredescribed in the literature where the temporary linkage is a liabileester bond. In theses cases, the functional group provided by thebioactive entity is either a hydroxyl group or a carboxylic acid (e.g.Y. Luo, M R Ziebell, G D Prestwich, “A Hyaluronic Acid—Taxol AntitumorBioconjugate Targeted to Cancer Cells”, Biomacromolecules 2000, 1,208-218, J Cheng et al, Synthesis of Linear, beta-Cyclodextrin BasedPolymers and Their Camptothecin Conjugates, Bioconjugate Chem. 2003, 14,1007-1017, R. Bhatt et al, Synthesis and in Vivo Antitumor Activity ofPoly(L-glutamic acid) Conjugates of 20(S)-Camptothecin, J. Med. Chem.2003, 46, 190-193; R. B. Greenwald, A. Pendri, C. D. Conover, H. Zhao,Y. H. Choe, A. Martinez, K. Shum, S. Guan, J. Med. Chem., 1999, 42,3657-3667; B. Testa, J. M: Mayer in Hydrolysis in Drug and ProdrugMetabolism, Wiley-VCH, 2003, Chapter 8).

Especially for therapeutic biomacromolecules but also for certain smallmolecule drugs, it may be desirable to link the macromolecular carrierto amino groups of the bioactive entity (i.e. N-terminus or lysine aminogroups of proteins). This will be the case if masking the drug'sbioactivity requires conjugation of a certain amino group of thebioactive entity, for instance an amino group located in an activecenter or a region or epitope involved in receptor binding. Also, duringpreparation of the prodrug, amino groups may be more chemoselectivelyaddressed and serve as a better handle for conjugating carrier and drugbecause of their greater nucleophilicity as compared to hydroxylic orphenolic groups. This is particularly true for proteins which maycontain a great variety of different reactive functionalities, wherenon-selective conjugation reactions lead to undesired product mixtureswhich require extensive characterization or purification and maydecrease reaction yield and therapeutic efficiency of the product.

Amide bonds as well as aliphatic carbamates are much more stable towardshydrolysis than ester bonds, and the rate of cleavage would be too slowfor therapeutic utility in a carrier-linked prodrug. Therefore it isadvantageous to add structural chemical components such as neighbouringgroups in order to exert control over the cleavability of the prodrugamide bond. Such additional cleavage-controlling chemical structuresthat are not provided by the carrier entity nor by the drug are calledlinker. Prodrug linkers can have a strong effect on the rate ofhydrolysis of a given temporary bond. Variation of the chemical natureof these linkers allows to engineer the linker properties to a greatextent.

For instance, prodrug linkers may be designed for enzyme-selectivity.Prerequisite for enzymatic dependence is that the linker structuredisplays a structural motif that is recognized as a substrate by acorresponding endogenous enzyme (FIG. 2).

Enzyme-catalyzed acceleration of prodrug cleavage is a desirable featurefor organ or cellular targeting applications. Targeted release of thebioactive entity is effected, if an enzyme, that selectively cleaves thelinkage, is specifically present in the organ or cell-type chosen fortreatment.

A typical property of an enzyme-dependent temporary linkage is itsstability with respect to hydrolysis. The temporary linkage itself willnot undergo autohydrolysis at a rate that would release drug to such anextent that a therapeutic effect could be induced in a normal dosingregime. It is only in the presence of the enzyme, that the attack of theenzyme on the linkage causes a significant acceleration of cleavage andconcomitant an enhancement of free drug concentration.

Several examples have been published for the prodrug activation ofamine-containing biologically active moieties by specific enzymes fortargeted release. In these cases, cleavage occurs in a one-step processwhich is catalyzed by the enzyme. G. Cavallaro et al., BioconjugateChem. 2001, 12, 143-151 describe the enzymatic release of an antitumoralagent by the protease plasmin. Cytarabin is coupled via the tripeptidesequence D-Val-Leu-Lys to the polymer alpha,beta-poly(N-hydroxyethyl)-DL-aspartamide (PHEA). Enzymatic release ofcytarabin is effected by the protease plasmin which concentration isrelatively high in various kinds of tumor mass.

Further examples for antitumoral polymeric prodrugs activated byspecific enzymes like beta lactamase (R. Satchi-Fainaro et al.,Bioconjugate Chem. 2003, 14, 797-804) and cysteine proteases likecathepsin B (R. Duncan et al. J. Contr. Release 2001, 74, 135-146) havebeen described. Wiwattanapatapee et al. (2003) outline a dendrimerprodrug for colonic delivery of 5-aminosalicylic acid. The drug moleculeis conjugated by an azo bond to “generation 3” PAMAM dendrimer.5-aminosalicylic acid is released in the colon by a bacterial enzymecalled azo reductase (W. R. Wiwattanapatapee, L. Lomlim, K. Saramunee,J. Controlled Release, 2003, 88: 1-9).

A. J. Garman et al. (A. J. Garman, S. B. Kalindjan, FEBS Lett. 1987, 223(2), 361-365 1987) use PEG5000-maleic anhydride for the reversiblemodification of amino groups in tissue-type plasminogen activator andurokinase. Regeneration of functional enzyme from PEG-uPA conjugate uponincubation at pH 7.4 buffer by cleavage of the maleamic acid linkagefollows first order kinetics with a half-life of 6.1 h. The prodrugcleavage was not investigated in the presence of enzymes, and it can beexpected—as explained above—that proteases present in the in vivoenvironment will significantly contribute to the cleavage of thetemporary amide linkage. A further disadvantage of this linkage is thelack of stability of the conjugate at lower pH values. This limits theapplicability of the linker to active agents which are stable at basicpH values, as purification of the active agent polymer conjugate has tobe performed under basic conditions to prevent premature prodrugcleavage.

Cascade mechanisms have proven particularly useful in the controlledrelease of drugs containing amino-group functionalities because linkercleavage characteristics can be optimized with greater flexibility thanin simple one-step prodrugs.

Cascade cleavage is enabled by linker compounds that are composed of astructural combination of a masking group and an activating group. Themasking group is attached to the activating group by means of a firsttemporary linkage such as an ester or a carbamate. The activating groupis attached to an amino-group of the drug molecule through a secondtemporary linkage, for instance a carbamate. The stability, orsusceptibility to hydrolysis of the second temporary linkage isdependent on the presence or absence of the masking group. In thepresence of the masking group, the second temporary linkage is highlystable and unlikely to release drug with therapeutically usefulkinetics. In the absence of the masking group, this linkage becomeshighly liabile, causing rapid cleavage and drug release.

Cleavage of the first temporary linkage is the rate-limiting step in thecascade mechanism. This first step may induce a molecular rearrangementof the activating group such as a 1,6-elimination. The rearrangementrenders the second temporary linkage so much more liabile that itscleavage is induced. Ideally, the cleavage rate of the first temporarylinkage is identical to the desired release rate for the drug moleculein a given therapeutic scenario. Furthermore, it is desirable thatcleavage of the second temporary linkage is instantaneous after itsliability has been induced by cleavage of the first temporary bond.

A variety of examples exist for cascade carrier prodrugs where themasking group functionality is performed by the carrier polymer itselfas shown diagrammatically in FIG. 3. In the systems discussed below, themasking group is not only part of the carrier but has also beenengineered for enzyme-dependence (FIG. 4). Only in the presence of acorresponding enzyme is the rate of cleavage of the first temporarylinkage sufficiently accelerated for therapeutic use.

R. B. Greenwald, A. Pendri, C. D. Conover, H. Zhao, Y. H. Choe, A.Martinez, K. Shum, S. Guan, J. Med. Chem., 1999, 42, 3657-3667 & PCTPatent Application WO-A-99/30727 describe a methodology for synthesizingpoly(ethylene glycol) prodrugs of amino-containing small moleculecompounds based on 1,4- or 1,6-benzyl elimination. In this approach,poly(ethylene glycol) as the polymeric carrier is attached to the benzylgroup by means of a first temporary linkage such as an ester, carbonate,carbamate, or amide bond. The benzyl group serves as the activatinggroup, and the PEG polymer also has the function of the masking group inthis cascade cleavage mechanism. The amino group of the drug molecule islinked via a second temporary linkage, containing a carbamate group, tothe benzyl moiety. The release of PEG from the drug molecule isinitiated by enzymatic cleavage of the first temporary linkage followedby a rapid 1,4- or 1,6-benzyl elimination, initiating cleavage of thesecond temporary linkage.

The same linker system is also used for releasable poly(ethylene glycol)conjugates of proteins (S. Lee, R. B. Greenwald et al. Bioconj. Chem.2001, 12 (2), 163-169). Lysozyme is used as model protein because itloses its activity when PEGylation takes place on the epsilon-aminogroup of lysine residues. Various amounts of PEG linker were conjugatedto the protein. Regeneration of native protein from the PEG conjugatesoccurrs by enzymatic cleavage in rat plasma or in non-physiological highpH buffer.

Greenwald et al. published in 2000 a poly(ethylene glycol) drug deliverysystem of amino-containing prodrugs based on trimethyl locklactonization (R. B. Greenwald et al. J. Med. Chem. 2000, 43(3),457-487; PCT Patent Application No. WO-A-02/089789). In this prodrugsystem, substituted o-hydroxyphenyl-dimethylpropionic acid is linked toPEG by an ester, carbonate, or carbamate group as a first temporarylinkage and to amino groups of drug molecules by means of an amide bondas second temporary linkage. The rate-determining step in drug releaseis the enzymatic cleavage of the first linkage. This step is followed byfast amide cleavage by lactonization, liberating a potentially toxicaromatic lactone side product.

Similar prodrug systems were described by F. M. H. DeGroot et al.(WO02083180 and WO04043493A1) and D. Shabat et al. (WO04019993A1).WO02083180 discloses a prodrug system with elongated and multiplelinkers based on 1,(4+2n) elimination. The masking moieties in theseexamples were specifically designed for enzymatic cleavage. Thisapproach was extended to dendritic prodrug system where one enzymaticactivating event triggered the release of more than one drug molecule(WO04043493A1). WO04019993A1 discloses a similar prodrug system based ona self-immolative dendrimer releasing many drug moieties upon a singleenzymatic activating event. These systems are characterized by theabsence of a polymeric carrier. Instead, oligomerization of prodruglinker components provides for a high molecular weight of the prodrug,and prodrug cleavage generates linker residues and free drug, but nopolymeric entity is released.

The disadvantage in the abovementioned prodrug systems described byGreenwald, DeGroot and Shabat is the release of potentially toxicaromatic small molecule side products like quinone methides aftercleavage of the temporary linkage. The potentially toxic entities arereleased in a 1:1 stoichiometry with the drug and can assume high invivo concentrations. This risk factor is even greater if self-immolativedendritic structures based on oligomers of the activating group areemployed and more aromatic side products than drug molecules arereleased.

More recently, R. B. Greenwald et al. (Greenwald et al. J. Med. Chem.2004, 47, 726-734) described a PEG prodrug system based onbis-(N-2-hydroxyethyl)glycin amide (bicin amide) linker. In this systemtwo PEG molecules are linked to a bicin molecule coupled to an aminogroup of the drug molecule. The first two steps in prodrug activation isthe enzymatic cleavage of both PEG molecules. Different linkages betweenPEG and bicin are described resulting in different prodrug activationkinetics. The main disadvantage of this system is the slow hydrolysisrate of bicin amide conjugated to the drug molecule (t1/2=3 h inphosphate buffer) which results in the release of a bicin-modifiedprodrug intermediate that may show different pharmacokinetic andpharmacodynamic properties as compared to the parent drug molecule.

Cascade prodrugs with masking groups that are part of the carrierpolymer are limited in the control of drug release kinetics. As maskinggroup cleavage is the rate-limiting step in the cascade mechanism, itsmolecular structure governs the kinetics. If the carrier polymer isidentical to the masking group, the structural flexibility is restrictedto the polymers' features. Alternatively, if the polymer requiresstructural modification in order to match the requirements forcontrolled cleavage, synthesis of corresponding structures may becomemore difficult. Also, the incorporation of masking group features into apolymer may change its safety profile.

Therefore is it preferred to structurally separate the masking group andthe carrier. This may be achieved by employing a permanent bond betweenpolymer carrier and activating group. This stable bond does notparticipate in the cascade cleavage mechanism. If the carrier is notserving as a masking group and the activating group is coupled to thecarrier by means of a stable bond, release of potentially toxic sideproducts such as the activating group is avoided. The stable attachmentof activating group and polymer also suppresses the release ofdrug-linker intermediates with undefined pharmacology.

Systems have been developed for targeted delivery of therapeutic agentsby rendering the masking group enzyme-dependent. Only in the presence ofa corresponding enzyme is the rate of cleavage of the first temporarylinkage connecting the masking group with the activating groupsufficiently accelerated for therapeutic use.

Antczak et al. (Bioorg Med Chem 9 (2001) 2843-48) describe a reagentwhich forms the basis for a macromolecular cascade prodrug system foramine-containing drug molecules. In this approach an antibody serves ascarrier, a stable bond connects the antibody to an activating moiety,carrying an enzymatically cleavable masking group. Upon enzymaticremoval of the ester-linked masking group, a second temporary bondcleaves and releases the drug compound, as shown in FIG. 6.

D. Shabat et al. (Chem. Eur. J. 2004, 10, 2626-2634) describe apolymeric prodrug system based on a mandelic acid activating moiety. Inthis system the masking group is linked to the activating moiety by acarbamate bond. The activating moiety is conjugated permanently to apolyacrylamide polymer via an amide bond. After enzymatic activation ofthe masking group by a catalytic antibody, the masking group is cleavedby cyclization and the drug is released. The activating moiety is stillconnected to the polyacrylamide polymer after drug release.

M.-R. Lee et al. describe (Angew. Chem. 2004, 116, 1707-1710) a similarprodrug system based on mandelic acid activating moiety and anenzymatically cleavable ester-linked masking group.

In all of these described prodrug-polymer systems the masking group isspecifically designed to be substrate to an enzyme, and masking groupcleavage will almost entirely depend upon enzymatic catalysis with thedisadvantages of interpatient variability, injection site variabilityand poor in vitro-in vivo correlation.

A major drawback of predominantly enzymatic cleavage is interpatientvariability. Enzyme levels may differ significantly between individualsresulting in biological variation of prodrug activation by enzymaticcleavage. Enyzme levels may also vary depending on the site ofadministration, for instance it is known that in the case ofsubcutaneous injection, certain areas of the body yield more predictabletherapeutic effects than others. To reduce this unpredictable effect,non-enzymatic cleavage or intramolecular catalysis is of particularinterest (see, for example, B. Testa, J. M: Mayer in Hydrolysis in Drugand Prodrug Metabolism, Wiley-VCH, 2003, page 5).

Furthermore, it is difficult to establish an in vivo-in vitrocorrelation of the pharmacokinetic properties for such enzyme-dependentcarrier-linked prodrugs. In the absence of a sound in vivo-in introcorrelation the optimization a release profile becomes a cumbersometask.

Also, the need for enzyme selectivity imposes a severe limitation on thestructural features that can be used in the prodrug linker. Thisrestriction greatly hinders the development of a soundstructure-activity relationship and consequently the optimization oflinker cleavage kinetics.

For these reasons, there is a need to provide novel linker and/orcarrier technologies for forming polymeric prodrugs of amine containingactive agents in order to overcome the limitations of the describedpolymeric prodrugs.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to polymeric prodrugs having temporarylinkages to amino groups of biologically active entities such aspeptides, proteins, natural products or synthetic chemical compounds.

The present invention addresses the disadvantages described above. Theinvention provides for polymeric cascade prodrugs characterized by amasking group containing a nucleophile and being distinct from thecarrier.

The nucleophile is in a suitable distance to a first temporary linkagewith an aromatic activating group capable of undergoing a 1,(4+2p)elimination reaction (with p=0, 1, 2, 3, 4, . . . ) after cleavage ofthe first temporary linkage. The invention is further characterized bythe activating group being connected to the amino group of a drugmolecule through a second temporary bond which is cleaved as aconsequence of the 1,(4+2p) elimination. An additional structuralfeature is the attachment of a polymeric carrier to the activating groupby means of a permanent bond.

The masking groups according to the present invention contain at leastone nucleophile Nu. This nucleophile, which can be for example aprimary, secondary or tertiary amino group can assist the cleavage ofthe masking group from the activating moiety by intramolecular catalysisor cyclization.

The invention provides for polymeric cascade prodrugs and correspondingpolymeric cascade prodrug linker reagents of Formula Ia or Ib.

wherein Y₁ to Y₅, R1 to R4, T, X, W, Nu and Ar are defined below:

Native drug release is effected by a two step mechanism. The first stepis the rate-determining cleavage of the first temporary linkage betweenthe masking group

and the activating moiety

from the polymeric prodrug in vivo.

As described above, cleavage of the masking group may be mediated by anenzymatic or a non-enzymatic step, such as pH-dependent hydrolysis orintramolecular cyclization. In the preferred embodiment of theinvention, cleavage is effected non-enzymatically by intramolecularcyclization or catalysis. The half-life of the cleavage kinetics in anaqueous buffer of pH 7.4 at 37° C. of the masking group according to thepresent invention is preferably between 1 hour and 6 months, morepreferably between 1 day and 3 months, and most preferably between 1 dayand 2 months.

The second and final step in release of the regenerated native drug isthe fast, spontaneous and irreversible so-called 1,4- or 1,6 or 1,(4+2p)(in which p=2, 3, 4 or higher) elimination of the

moiety of the remaining polymeric prodrug of formula Ia or formula Ib,respectively.

This mechanism of native drug release from a polymeric prodrug triggeredby hydrolytic cleavage of the masking group followed by a1,6-elimination step of the activating group is exemplified by apolymeric prodrug according to the present invention.

Definition of Y₁ to Y₅, R1 to R4, T, X, W, Nu and Ar in formula Ia or Ib

T is D or A

In the case where the inventive structure is a polymeric cascade prodruglinker reagent, T is A, and A is a leaving group. Non-limiting examplesof suitable leaving groups A include but are not limited to chloride,bromide, fluoride, nitrophenoxy, imidazolyl, N-hydroxysuccinimidyl,N-hydroxybenzotriazolyl, N-hydroxyazobenzotriazolyl, pentafluorphenoxy,N-hydroxysulfosuccinimidyl, or any other leaving group known by thoseskilled in the art.

In the case where the inventive structure is a polymeric cascadeprodrug, T is D, and D is a residue of an amine-containing biologicallyactive material including but not limited to small molecule bioactiveagents or biopolymers like proteins, polypeptides and oligonucleotides(RNA, DNA), peptide nucleic acids (PNA),

Note that in this description reference is often made to prodrugs. Atrue prodrug is found when T is the residue of the amine-containingbiologically active material or moiety. If T is a leaving group A thenthe formula represents a polymeric cascade prodrug linker reagent. Forsimplicity these will be referred to prodrugs in this description. Itwill be understood from the context whether a true prodrug or a reagentas a precursor is meant.

DETAILED DESCRIPTION OF THE INVENTION

Suitable organic small molecule bioactive moieties include, withoutlimitation, moieties such as central nervous system-active agents,anti-infective, anti-neoplastic, antibacterial, anti-fungal, analgesic,contraceptive, anti-inflammatory, steroidal, vasodilating,vasoconstricting, and cardiovascular agents with at least one primary orsecondary amino group. Non-exclusive examples of such compounds aredaunorubicin, doxorubicin, idarubicin, mitoxantron, aminoglutethimide,amantadine, diaphenylsulfon, ethambutol, sulfadiazin, sulfamerazin,sulfamethoxazol, sulfalen, clinafloxacin, moxifloxacin, ciprofloxaxin,enoxacin, norfloxacin, neomycin B, sprectinomycin, kanamycin A,meropenem, dopamin, dobutamin, lisinopril, serotonin, carbutamid,acivicin, etc.

Suitable proteins and polypeptides having at least one free amino groupinclude but are not limited to ACTH, adenosine deaminase, agalsidase,albumin, alfa-1 antitrypsin (AAT), alfa-1 proteinase inhibitor (API),alteplase, anistreplase, ancrod serine protease, antibodies (monoclonalor polyclonal, and fragments or fusions), antithrombin III,antitrypsins, aprotinin, asparaginases, biphalin, bone-morphogenicproteins, calcitonin (salmon), collagenase, DNase, endorphins,enfuvirtide, enkephalins, erythropoietins, factor VIIa, factor VIII,factor VIIIa, factor IX, fibrinolysin, fusion proteins,follicle-stimulating hormones, granulocyte colony stimulating factor(G-CSF), galactosidase, glucagon, glucagon-like peptides like GLP-1,glucocerebrosidase, granulocyte macrophage colony stimulating factor(GM-CSF), phospholipase-activating protein (PLAP), gonadotropinchorionic (hCG), hemoglobins, hepatitis B vaccines, hirudin,hyaluronidases, idurnonidase, immune globulins, influenza vaccines,interleukins (1 alfa, 1 beta, 2, 3, 4, 6, 10, 11, 12), IL-1 receptorantagonist (rhIL-1ra), insulins, interferons (alfa 2a, alfa 2b, alfa 2c,beta 1a, beta 1b, gamma 1a, gamma 1b), keratinocyte growth factor (KGF),transforming growth factors, lactase, leuprolide, levothyroxine,luteinizing hormone, lyme vaccine, natriuretic peptide, pancrelipase,papain, parathyroid hormone, PD GF, pepsin, platelet activating factoracetylhydrolase (PAF-AH), prolactin, protein C, octreotide, secretin,sermorelin, superoxide dismutase (SOD), somatropins (growth hormone),somatostatin, streptokinase, sucrase, tetanus toxin fragment, tilactase,thrombins, thymosin, thyroid stimulating hormone, thyrotropin, tumornecrosis factor (TNF), TNF receptor-IgG Fc, tissue plasminogen activator(tPA), TSH, urate oxidase, urokinase, vaccines, plant proteins such aslectins and ricins.

Also included herein is any synthetic polypeptide or any portion of apolypeptide with in vivo bioactivity. Furthermore, proteins prepared byrecombinant DNA methodologies including mutant versions ofaforementioned proteins, antibody fragments, single chain bindingproteins, catalytic antibodies and fusion proteins are included.

Preferred proteins are antibodies, calcitonin, G-CSF, GM-CSF,erythropoietins, hemoglobins, interleukins, insulins, interferons, SOD,somatropin, TNF, TNF-receptor-IgG Fc, and GLP-1.

X is a spacer moiety such as R5-Y6

Y₁, Y₂ can each be either O, S, or NR6, independently of each other.

Y₃, Y₅ can each be either O or S, independently of each other.

Y₄ is O, NR6, or —C(R7)(R8)—

Y₆ is O, S, NR6, succinimide, maleimide, unsaturated carbon-carbon bondsor any heteroatom containing a free electron pair, or is not present.

R2 and R3 are selected independently from hydrogen, substituted ornon-substituted linear, branched or cyclical alkyl or heteroalkyl,aryls, substituted aryls, substituted or non-substituted heteroaryls,cyano, nitro, halogen, carboxy, carboxyalkyl, alkylcarbonyl,carboxamidoalkyl, etc.

The term “heteroalkyl” in the context of the present invention denotes(linear, cyclical or branched) alkyl chains where the alkyl chainscontain or are substituted with at any position one or more heteroatoms,selected independently from O, S, N, P, Si, Cl, F, Br, I, etc. orgroups, selected independently from carboxamide, carboxylic ester,phosphonate ester, phosphate ester, double or triple bonds, carbamate,urea, thiourea, thiocarbamate, oxime, cyano, carboxyl, carbonyl, etc.

Each R4 substitution on Ar may be the same or different and is selectedindependently from hydrogen, substituted or non-substituted linear,branched or cyclical alkyl or heteroalkyl, aryl, substituted aryl,substituted or non-substituted heteroaryl, substituted ornon-substituted linear, branched, or cyclical alkoxy, substituted ornon-substituted linear, branched, or cyclical heteroalkyloxy, aryloxy,heteroaryloxy, cyano, halogen, etc.

R4 is selected preferably from small substituents such as hydrogen,methyl, ethyl, ethoxy, methoxy, and other C1 to C6 linear, cyclical orbranched alkyls and heteroalkyls.

n is zero or a positive integer.

R7 and R8 are selected independently from hydrogen, substituted ornon-substituted linear, branched or cyclical alkyl or heteroalkyl,aryls, substituted aryls, substituted or non-substituted heteroaryls,cyano, halogen, etc.

R5 is selected from substituted or non-substituted linear, branched orcyclical alkyl or heteroalkyl, aryls, substituted aryls, substituted ornon-substituted heteroaryls, etc.

R6 is selected from hydrogen, substituted or non-substituted linear,branched or cyclical alkyl or heteroalkyl, aryls, substituted aryls,substituted or non-substituted heteroaryls, etc.

R1 is a polymer.

Non-limiting examples for suitable polymers are polyalkyloxy-basedpolymers like poly(propylene glycol) or poly(ethylene glycol), dextran,chitosan, hyaluronic acid and derivatives, alginate, xylan, mannan,carrageenan, agarose, cellulose, starch, hydroxyethyl starch (HES) andother carbohydrate-based polymers, poly(vinyl alcohols),poly(oxazolines), poly(anhydrides), poly(ortho esters),poly(carbonates), poly(urethanes), poly(acrylic acids),poly(acrylamides) such as poly(hydroxypropylmethacrylamide) (HMPA),poly(acrylates), poly(methacrylates) likepoly(hydroxyethylmethacrylate), poly(organophosphazenes),poly(siloxanes), poly(vinylpyrrolidone), poly(cyanoacrylates),poly(esters) such as poly(lactic acid) or poly(glycolic acids),poly(iminocarbonates), poly(amino acids) such as poly(glutamic acid),collagen, gelatin, copolymers, grafted copolymers, cross-linkedpolymers, hydrogels, and block copolymers from the above listedpolymers.

Hydrogels may be defined as three-dimensional, hydrophilic oramphiphilic polymeric networks imbibing large quantities of water. Thenetworks are composed of homopolymers or copolymers, are insoluble dueto the presence of covalent chemical or physical (ionic, hydrophobicinteractions, entanglements) crosslinks. The crosslinks provide thenetwork structure and physical integrity. Hydrogels exhibit athermodynamic compatibility with water which allows them to swell inaqueous media.(see.: N. A. Peppas, P. Bures, W. Leobandung, H. Ichikawa,Hydrogels in pharmaceutical formulations, Eur. J. Pharm. Biopharm. 2000,50, 27-46). The chains of the network are connected in such a fashionthat pores exist and that a substantial fraction of these pores are ofdimensions of between 1 and 1000 nm. By selecting certain polymerizationconditions, the hydrogel may be obtained in the form of an amorphous gelor as beaded resin. Such soft beads may have a diameter of between 1 and1000 micrometer.

Hydrogels may be synthesized from the polymers and copolymers listedabove and physically cross-linked or chemically cross-linked by radical,anionic or cationic polymerization, by chemical reactions likecondensation or addition reactions as described in W. E. Hennink and C.F. van Nostrum, Adv. Drug Del. Rev. 2002, 54, 13-36.

Further examples include branched and hyperbranched polymers. Examplesfor such polymers include dendrimers and other dense star polymers. (R.Esfand, D. A. Tomalia, Drug Discov Today, 2001, 6(8), 427-436; P. M.Heegaard, U. Boas, Chem. Soc. Rev. 2004 (33(1), 43-63; S. M. Grayson, J.M. Frechet, Chem. Rev. 2001, 101 (12), 3819-3868).

R1 can also be a biopolymer like a protein. Non-limiting examples ofsuch polymers include albumin, antibodies, fibrin, casein, and otherplasma proteins.

Each R1 polymer can carry one or more biologically active substanceslinked to the polymer by conjugation with a second prodrug linker asdescribed herein or any other linker known to the person skilled in theart. The polymers may have further substituents and may befunctionalized for attachment to the spacer moiety X. Non-limitingexamples of such functional groups comprise carboxylic acid andactivated derivatives, amino, maleimide, thiol, sulfonic acid andderivatives, carbonate and derivatives, carbamate and derivatives,hydroxyl, aldehyde, ketone, hydrazine, isocyanate, isothiocyanate,phosphoric acid and derivatives, phosphonic acid and derivatives,haloacetyl, alkyl halides, acryloyl, arylating agents like arylfluorides, hydroxylamine, disulfides like pyridyl disulfide, vinylsulfone, vinyl ketone, diazoalkanes, diazoacetyl compounds, epoxide,oxirane, and aziridine.

Preferred functional groups for the R1 polymer include but are notlimited to thiol, maleimide, amino, carboxylic acid and derivatives,carbonate and derivatives, carbamate and derivatives, aldehyde, andhaloacetyl.

Especially preferred functional groups include thiol, maleimide, amino,carboxylic acid and derivatives, carbamate and derivatives, andcarbonate and derivatives thereof.

Non-limiting examples for suitable bonds or groups formed between X andR1 include disulfide, S-succinimido, amide, amino, carboxylic ester,sulfonamide, carbamate, carbonate, ether, oxime, hydrazone, urea,thiourea, phosphate, phosphonate, etc.

Preferred bonds or groups formed between X and R1 compriseS-succinimido, amide, carbamate, and urea.

Preferably, the R1 polymers are well hydrated, degradable or excretable,nontoxic and non-immunogenic in mammals. Preferred R1 polymers includepolyalkoxy-based polymers like polyethylene glycol and polyethyleneglycol reagents as those described in Nektar Inc. 2003 catalog “NektarMolecule Engineering—Polyethylene Glycol and Derivatives for AdvancedPEGylation” and branched, hyperbranched, cross-linked polymers andhydrogels, and proteins like albumin.

W is selected from substituted or non-substituted linear, branched orcyclical alkyl, aryls, substituted aryls, substituted or non-substitutedlinear, branched or cyclical heteroalkyl, substituted or nonsubstitutedheteroaryls, etc.

W is selected preferably from non-toxic substituted or non-substitutedlinear, branched or cyclical alkyls or heteroalkyls.

Preferred variations of

Especially preferred variations of

forming polymeric prodrugs of the following formulas:

In formula Iaa and Iba R6 may also be Nu-W.

At least one Nu is present in Nu-W.

Nu is a nucleophile that can perform a nucleophilic attack at thecarbonyl carbon of

and thus catalyse the cleavage of the masking group by intramolecularcatalysis or cyclization (FIG. 8). FIG. 8 shows an example according toformula Ia or Ib wherein the cleavage of the masking group is byintramolecular cyclization. In cases where Nu only catalyses thecleavage of the masking group by intramolecular catalysis, no cyclicalproduct of the masking group is formed.

Preferred nucleophiles include primary, secondary and tertiary aminogroups, thiol, carboxylic acid, hydroxylamine, hydrazine, and nitrogencontaining heteroaryl. Especially preferred nucleophiles includeprimary, secondary and tertiary amino groups. In order to effectivelycatalyse the cleavage of the masking group, the spacing between thenucleophile Nu and Y₂ is preferably between three and fifteen atoms.More preferably, the spacing between Nu and Y₂ is between four and tenatoms. The at least one nucleophile Nu may be attached anywhere to W(e.g. at the terminus or in the middle of W) or may be part of W.

Preferred variations for the masking group

are selected independently from

where

forms a primary, secondary or tertiary amine nucleophile Nu.

These preferred variations result in polymeric prodrugs according to thefollowing formulas:

R9, R10 are selected independently from hydrogen, substituted ornon-substituted alkyl or heteroalkyl, substituted or non-substitutedaryl or heteroaryl.

Especially preferred variations for the masking group

are selected from

Preferably, R9, R10, R11 and R12 are selected independently fromhydrogen, substituted or non-substituted alkyl and R7 and/or R8 are nothydrogen.

R6 may also be

and is preferably not hydrogen.

Surprisingly it was found, that the masking group can modifyirreversibly the amine containing biologically active moiety when thenucleophile Nu is absent in the masking group. As shown in the examplesection, during release of the bioactive moiety insulin from a polymericprodrug with a pentanoyl masking group which is not part of the presentinvention (as it does not contain a nucleophile), approximately 30% ofthe insulin molecule was modified with the masking group by acyltransfer. The mechanism of an example of this modification where Dcontains an additional free amino group that serve as nucleophile foracyl transfer from the masking group is shown in FIG. 9.

Ar of formula Ia or Ib is a multi-substituted aromatic hydrocarbon or amulti-substituted aromatic heterocycle. To be aromatic, the number of pielectrons must satisfy the Hückel rule (4n+2) and the cycle has to beplanar. A huge variety of compounds satisfy these criteria and thus aresuitable as Ar in formula Ia or Ib. Non-limiting preferred aromaticmoieties include:

wherein W is O, N, or S, independent from each other.

Y₂ and

in formula Ia or Y₂ and

in formula Ib have to be arranged on the aromatic ring in such a fashionthat a 1,4- or 1,6- or 1,(4+2p), with p=2, 3, 4 and higher, eliminationcan take place (see above). For example, in the case of a 6-memberedring, the substituents have to be arranged ortho or para.

Preferred moieties for Ar are mono- and dicyclic aromatic hydrocarbonsor aromatic heterocycles.

Especially preferred moieties are monocyclic five- or six-memberedaromatic hydrocarbons or aromatic heterocycles.

General Synthesis Procedures of the Polymeric Prodrugs

Synthesis of representative examples of polymeric prodrugs according tothe present invention is described in the Examples section.

Prodrugs of the present invention can be prepared in various differentfashions. FIG. 10 shows general routes for the synthesis of thepolymeric prodrugs of the present invention according to formula Ia.

In a first method, intermediate (III) is provided by acylating Y₂ ofstarting material (II) with the masking group

For this, X or Nu may have to be protected with a reversible protectinggroup PG₁. Suitable protecting groups are described in T W Greene, P. G.M. Wuts, Protective groups in organic synthesis, 1999, John Wiley &Sons, 3^(rd) ed. From intermediate (III) two alternative routes can beused to yield (Iaa). In a first route intermediate (III) is activated byan activating agent such as 4-nitrophenyl chloroformate or disuccinylcarbonate to yield (IV). The amine containing drug molecule is attachedto (IV) to yield (V) by displacing the leaving group of the activatedintermediate (IV). After deprotection of X such as by treatingintermediate (V) with reagents like trifluoroacetic acid or DTT (whereapplicable) deprotected intermediate (V) is then reacted with polymer R1to yield the polymeric prodrug (Iaa).

In a second route the polymer R1 is first attached to the intermediate(III) after deprotection of X (where applicable) to form intermediate(VI). After an activation step intermediate (VII) is formed. (VII) isreacted with the amine containing drug molecule to form the polymericprodrug (Iac).

In a second method, intermediate (VIII) is provided by activatingstarting material (II) by an activating agent such as 4-nitrophenylchlorofomate. For this, Y₂ and/or X may have to be protected with aprotecting group PG₂ and/or PG₁. Amine containing drug is reacted withintermediate (VIII) to form (IX). In a first route, Y₂ of (IX) isselectively deprotected and acylated to form intermediate (V) which isfurther processed to (Iaa) as described above. In a second route X isselectively deprotected and reacted with polymer R1 to form intermediate(X). Y₂ of (X) is then deprotected and acylated to form the polymericprodrug (Iac).

In a third method starting material (II) is reacted with polymer R1 toform intermediate (XI). In one route, intermediate (XI) can be acylatedto form intermediate (VI) which processed as described above to formpolymeric prodrug (Iaa). In a second route, Y₂ is protected by theprotecting group PG₂, activated and reacted with the amine containingdrug molecule to form (X). Intermediate (X) is then processed asdescribed above to form the polymeric prodrug (Iac).

For all methods described, further functional groups such as Y₃ ornucleophiles present in Nu-W may have to be protected with suitableprotecting groups.

Polymeric prodrugs according to formula Ib can be prepared by methodsdescribed above for prodrugs according to formula Ia using startingmaterial IIb instead of II in FIG. 10.

It is understood, that linker structures according to the outlinedinvention and carrying protecting groups or leaving groups as describedand used in the synthesis of corresponding polymeric prodrugs areconsidered within the range of the invention.

Application of the Polymeric Prodrugs in Molecular Therapy

For polymeric cascade prodrugs it is desirable for the cleavage kineticsof the first temporary linkage to proceed under conditions present inthe blood circulation of the human body (pH 7.4, 37° C.). Mostimportantly, cleavage of the first temporary linkage should be based onhydrolysis and exhibit none or only very limited dependence uponchemical or biochemical or physicochemical entities present in the humanblood circulation such as enzymes, salts or binding proteins.

It was now surprisingly found that the rate of cleavage of the firsttemporary linkage connecting the masking group with the activating groupas well as its dependence upon blood components can be controlled byneighbouring group effects mediated by nucleophilic functional groups(such as primary, secondary or tertiary amines) present in the maskinggroup and positioned in a distance to the corresponding temporary bond.If the masking group is structured in this fashion, an intramolecularreaction with contribution of the nucleophile governs the kinetics ofthe linkage (FIG. 5 and FIG. 8).

Key advantage of the polymeric prodrugs of the present invention istheir predominantly non-enzymatic cleavage: the half-life of the prodrugin suitably buffered human blood plasma of pH 7.4 (with aqueous bufferconcentration <50%) is at least 50% of the half-life of the prodrug inenzyme-free buffer pH 7.4.

This feature allows for better predictability and control of releaserates after administration to a living organism and reduces interpatientvariability.

In contrast to the enzymatic dependency of masking group removal asdescribed in abovementioned examples of Antzczak et al., Shabat et al.and Lee et al., a higher level of control over release rates can beachieved if the masking group has enzyme-independent, self-eliminatingproperties.

The masking groups according to the present invention contain at leastone nucleophile Nu. Structural features of this masking group such asnucleophilicity of the amine group and ring-forming capacity may besystematically optimized in order to precisely adjust the rate ofprodrug cleavage. Such intramolecular reactions resulting in unmaskingand subsequent rearrangement are highly independent from enzymes due tothe fact that intramolecular reactions are generally preferred overintermolecular reactions as shown diagrammatically in FIG. 8.

In another embodiment of the invention, independency of prodrug cleavagefrom enzyme levels is achieved by providing a prodrug containing asterically demanding carrier group as is shown in FIG. 7.

Such encapsulation or sterical protection by the sterically demandingcarrier group may be conferred by a branched, hyperbranched, crosslinkedor self-assembled structure of the carrier polymer. Such polymers tendto form a densely packed molecular volume, as exemplified for instancein dendrimers, dense star polymers or bead-shaped nano- andmicroparticles or amorphous gels. If the linkage of the polymer carrierto the drug is located in the interior of the polymer carrier, thelinked drug will be efficiently encapsulated and protected fromenzymatic attack. In this case, sterical hindrance by the polymerprevents enzymes from accessing and cleaving the temporary linkages.

In yet another embodiment, enzyme-independent prodrug cleavage isachieved by combining an intramolecular self-eliminating masking groupwith an encapsulating hyperbranched or crosslinked or self-assembledcarrier.

A further advantage of the present invention is the release of anunmodified biologically active moiety. In cases where the biologicallyactive moiety contains further reactive functional groups like aminogroups of lysine residues in proteins, an unwanted side reaction betweenthe masking group and the biologically active moiety can occur. Thereactive functional groups of the biologically active moiety may reactwith the masking group, forming a stable covalent bond and resulting inthe release of a modified biologically active moiety. This potentialside reaction is shown schematically in FIG. 9. The occurrence of suchside reactions is shown in the examples section using polymeric prodrugswhich are not part of the present invention with simple masking groupslike a pentanoyl residue without a nucleophile Nu present in the maskinggroup and as described by Antczak et al. or Lee et al. The side reactionin this linker system is suppressed using polymeric prodrugs accordingto the present invention with intramolecularly activated masking groupsthat contain nucleophiles Nu (see example section).

Enzyme-independent release control enables depot formulations withoutthe need for encapsulation. Until now, many biocompatible materials likehydrogels with large pore sizes could not be used for depot formulationsdue to their lack of encapsulation properties. From such well-hydratedand mechanically soft biocompatible materials, biologically activemoiety would be released too fast for most therapeutic applications. Incombination with the prodrug linkers described in this invention, thecarrier material may be optimized for its biocompatibility properties asthe release is solely governed by the linker cleavage kinetics and doesnot require chemical or enzymatic degradation of the polymer carrieritself.

Release rates are governed by a substantially non-enzymatic chemicalreaction which is in turn dependent on the molecular structure of thelinker. Systematic or random modifications of the chemical structure,for instance by changing substituents in one or more positions, forinstance a masking group in a cascade prodrug, allows for the generationof prodrug linkers with differing release rates. It is thereforepossible to create a variety of prodrug linkers and select those fast orslow cleaving prodrug linkers according to the demands posed by a givenmedicinal or therapeutic application.

Another advantageous feature which is part of this invention is theattachment of the polymer carrier through a stable covalent bond to anactivating moiety involved in a double or cascade prodrug releasemechanism. As part of this invention, the activating moiety remainsattached to the polymer carrier after drug release and therefore cannotdiffuse into the environment. Permanent linkage of the polymer carrierto the activating group greatly reduces the any side-reactivity of theactivating moiety and the probability of unwanted toxic effects. Inother polymeric cascade prodrugs known in the art, the activating moietyis released in addition to the drug. Due to the molecular rearrangementmechanisms used in cascade prodrugs, the activating moiety is releasedin a highly reactive form and may cause direct damage to surroundingbiomolecules, or potentially toxic derivatives of the activating moietymay be formed in vivo.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a carrier-linked prodrug.

FIG. 2 shows an enzyme-dependent carrier-linked prodrug.

FIG. 3 shows a cascade prodrug where the masking group is part of thecarrier.

FIG. 4 shows an enzyme-dependent cascade prodrug where the masking groupis part of the carrier.

FIG. 5 shows a self-cleaving cascade prodrug where the masking group isseparate from the carrier.

FIG. 6 shows an enzyme-dependent cascade prodrug where the masking groupis separate from the carrier.

FIG. 7 shows a cascade prodrug where the carrier is stericallyprotecting the masking group.

FIG. 8 shows cleavage of the masking group by intramolecularcyclisation.

FIG. 9 shows a possible side reaction of polymeric prodrug activation.

FIG. 10 shows general synthesis methods.

FIG. 11 shows mass spectra of prodrug released insulin molecules.

EXAMPLE

Materials

Fmoc-amino acids, resins and PyBOP were purchased from Novabiochem andare named according to the catalogue. Fmoc-Ado-OH was obtained fromNeosystem. All additional chemicals were purchased from Sigma Aldrich.Recombinant human insulin was from ICN Biomedicals (USA).Maleimide-PEG5k was obtained from Nektar (USA).5-(and-6)-carboxyfluorescein succinimidyl ester (mixed isomers) wasobtained from Molecular Probes.

Solid Phase Synthesis Reaction Medium

Solid phase synthesis was performed on NovaSyn TG Sieber amide resinwith a loading of 0.17 mmol/g or 2-chlorotrityl chloride resin with aloading of 1.4 mmol/g. Syringes equipped with polypropylene frits wereused as reaction vessels.

Standard Coupling Cycle for Fmoc-Protected Amino Acids

For fmoc protecting-group removal, the resin was repeatedly (threetimes, 4 min each) agitated with 2/2/96 (v/v/v) piperidine/DBU/DMF andrepeatedly (six times) washed with DMF.

Coupling of fmoc-protected amino acids to free amino groups on resin wasachieved by agitating the resin with 3 equivalents (eq) of fmoc-aminoacid, 3 eq PyBOP and 6 eq DIEA in relation to free amino groups in DMFfor 60 min.

Finally, the resin was repeatedly (five times) washed with DMF.

Standard Cleavage Protocol for TentaGel Sieber Amide Resin

Upon completed synthesis, the resin was washed with DCM, dried in vacuoand treated repeatedly (five times) with 97/2/1 (v/v) DCM/TES/TFA. Afterevaporation, compounds were purified by preparative RP-HPLC (Waters600).

Standard Cleavage Protocol for 2-chlorotrityl Chloride Resin

Upon completed synthesis, the resin was washed with DCM, dried in vacuoand treated two times for 30 minutes with 65/35 (v/v) HFIP/DCM. Aftercombining the eluates the volatile components were evaporated.

Analysis

Mass spectrometry (MS) was performed on a Waters ZQ 4000 ESI instrumentand spectra were, if necessary, interpreted by Waters software MaxEnt.

Size exclusion chromatography was performed using an Amersham BioscienceAEKTAbasic system equipped with a Superdex 200 column (AmershamBioscience). NMR spectra were recorded on a Bruker AC300.

Overview—Synthesis of Polymeric Prodrugs According to Formula Ia withEster-Linked Masking Groups

Synthesis of Polymeric Prodrugs According to Formula Ia withCarbamate-Linked Masking Groups

Synthesis of Polymeric Prodrugs According to Formula Ib with aCarbamate-Linked Masking Group

Synthesis of Compound 2

Mmt-chloride (1 eq) and mercaptopropionic acid (1.1 eq) were dissolvedin TFA and incubated for 30 min. The solvent was removed under reducedpressure. The product was dissolved in pyridine, diluted in water,acidified by acetic acid and extracted with ether. The ether phase wasseparated and dried over Na₂SO₄. Solvent was removed under reducedpressure and product 2 was purified by RP-HPLC.

Synthesis of Compounds 3a and 3b

3a R1=H

3b R1=OMe

Octopamine hydrochloride (1a) (2 eq), DIEA (4 eq), and PyBOP (1 eq) weredissolved in DMF, 2 (1 eq) was added and the mixture was reacted for 50min at room temperature. After addition of acetic acid (7 eq) product 3awas purified by RP-HPLC.

3b was synthesized from normetanephrine hydrochloride (1b) as describedabove.

3a: MS [M+Na]⁺=536 (MW+Na calculated=536.2 g/mol)

3b: MS [M+Na]⁺=566 (MW+Na calculated=566.2 g/mol)

Synthesis of Mercaptothiazolide 4

2-Mercaptothiazoline and triethylamine (1.5 eq) were dissolved in dryTHF and pentanoyl chloride (1 eq) was added. The mixture was stirred for1 h at 50° C. under inert gas atmosphere and was allowed to cool to roomtemperature. 0.5 N aqueous HCl was added and the separated organicphases were dried over Na₂SO₄. After concentration in vacuo the residuewas purified by silica gel column chromatography using heptane/ethylacetate (1/1) as mobile phase. Mercaptothiazolide 4 was collected as aviscous yellow oil.

4 R_(f) (heptane/ethyl acetate 1:1)=0.7

Synthesis of Compounds 5a and 5b

General Synthesis Protocol:

1 g 2-chlorotrityl chloride resin (loading 1.6 mmol/g) was incubated for1 h with 850 mg (2.4 mmol) Fmoc-Ile-OH and 840 μl (4.8 mmol) DIEA in 5ml 1/1 DCM/DMF. After fmoc removal and washing of the resin with DMF,boc-aminobutyric acid was coupled to 0.5 g resin according to thestandard coupling method. Compound 5a was cleaved from the resin with97/1/2 (v/v) DCM/TFA/TES for 45 min. After neutralisation with pyridine,solvents were removed under reduced pressure and 5a was purified byRP-HPLC.

5b was synthesized from boc-aminohexanoic acid as described above.

5a MS [M+Na]⁺=339.2 (MW+Na calculated=339.4 g/mol)

5b MS [M+Na]⁺=367.4 (MW+Na calculated=367.5 g/mol)

Synthesis of Compound 6a

Mercaptothiazolide 4 (1 eq), phenol 3a (4 eq) and DMAP (4 eq) wererefluxed in DCM for 2 h under nitrogen atmosphere. After neutralizationwith acetic acid, the solvent was removed in vacuo and product 6a waspurified by RP-HPLC.

6a MS [M+Na]⁺=620 (MW+Na calculated=620.3 g/mol)

Synthesis of Compounds 6b to 6e

General Synthesis Protocol:

Carboxylic acid 5a (1 eq), phenol 3b (1 eq), DIC (1 eq), and DMAP (2 eq)in DMF were reacted for 1 h at room temperature. After addition ofacetic acid (4 eq) the resulting carboxylic ester 6c was purified byRP-HPLC.

6d was synthesized as described above using 5b and 3b as startingmaterials.

6b was synthesized as described above using Z-Lys(Boc)-OH and 3b.

6e was synthesized as described above using 5b and 3a.

6b MS [M+Na]⁺=928 (MW+Na calculated=928.6 g/mol)

6c MS [M+Na]⁺=864 (MW+Na calculated=864.5 g/mol)

6d MS [M+Na]⁺=892 (MW+Na calculated=892.6 g/mol)

6e MS [M+Na]⁺=862 (MW+Na calculated=862.6 g/mol)

Synthesis of Compounds 7a to 7e

General Synthesis Protocol:

Alcohol 6a (1 eq), 4-nitrophenyl chloroformate (10 eq), and DIEA (10 eq)were stirred in dry dioxane for 3 h at room temperature under nitrogenatmosphere. After addition of acetic acid (25 eq) the mixtures werediluted with 7/3 (v/v) acetonitrile/H₂O and the carbonate 7a waspurified by RP-HPLC.

7b, 7c, 7d, or 7e were synthesized from 6b, 6e, 6d, or 6e, respectively,as described above.

7a MS [M+Na]⁺=785 (MW+Na calculated=785.5 g/mol)

7b MS [M+Na]⁺=1093 (MW+Na calculated=1093.7 g/mol)

7c MS [M+Na]⁺=1029 (MW+Na calculated=1029.6 g/mol)

7d MS [M+Na]⁺=1057 (MW+Na calculated=1057.6 g/mol)

7e MS [M+Na]⁺=1027 (MW+Na calculated=1027.6 g/mol)

Synthesis of Compounds 8a to 8c (N^(αA1)-Linker-Insulin)

General Synthesis Protocol:

Rh-Insulin in 1/1 (v/v) DMSO/DMF was mixed with a solution of 0.9 eqcarbonate 7a in DMSO. The resulting solution was adjusted to basic pHwith DIEA and stirred for 1.5 h at RT. RP-HPLC purification gaveMmt-protected intermediate.

After lyophilization, the Mmt-protected intermediate was mixed with 95:5(v/v) TFA/triethylsilane and stirred for 5 min. Volatiles were removedunder nitrogen flow and 8a was purified by RP-HPLC and lyophilized.Regioselectivity of insulin modification was verified by DTT reductionand MS analysis.

8b or 8c were synthesized from 7c, or 7d, respectively, as describedabove.

8a MS [M+2H]²⁺=3078.9; [M+3H]³⁺=2053.2 [M+4H]⁴⁺=1540.6 (MWcalculated=6158 g/mol)

8b MS [M+2H]²⁺=3152.9; [M+3H]³⁺=2100.6 [M+4H]⁴⁺=1575.8 (MWcalculated=6302 g/mol)

8c MS: [M+3H]³⁺=2110.7; [M+4H]⁴⁺=1583.7; [M+5H]⁵⁺=1266.6 (MWcalculated=6330 g/mol)

Synthesis of Compounds 8d to 8g(N^(εB29)-Fluorescein-N^(αA1)-Linker-Insulin)

Synthesis of N^(εB29)-Fluorescein Insulin:

80 mg (13.8 μmol) rh-insulin were dissolved in 4 ml 1/1 (v/v) DMF/DMSOand 40 μl DIEA were added. 8 mg (17 μmol) 5-(and-6)-carboxyfluoresceinsuccinimidyl ester were added and the solution was stirred for 30 min atroom temperature. 4 ml 5/5/1 (v/v/v) acetonitrile/water/acetic acid wereadded, product N^(εB29)-fluorescein insulin was purified by RP-HPLC andlyophilized. The conjugation site was verified by reduction ofN^(εB29)-fluorescein insulin with 1,4-dithiothreitol, protease digestionand MS analysis.

MS: [M+2H]²⁺=3084.0; [M+3H]³⁺=2054.6 (MW calculated=6166 g/mol)

N^(εB29)-fluorescein insulin in 1/1 (v/v) DMF/DMSO was mixed with asolution of 0.9 eq carbonate 7b in DMSO. The resulting solution wasadjusted to basic pH with DIEA and stirred for 3 h at RT. RP-HPLCpurification gave Mmt-protected intermediate.

After lyophilization, the intermediate was dissolved in 95/5 (v/v)TFA/triethylsilane and stirred for 5 min. Volatiles were removed undernitrogen flow and 8d was purified by RP-HPLC and lyophilized.

8e, 8f, or 8g were synthesized as described above using 7c, 7d, or 7e,respectively.

8d MS: [M+2H]²⁺=3364.1; [M+3H]³⁺=2242.7; [M+4H]⁴⁺=1681.5 (MWcalculated=6724 g/mol)

8e MS: [M+3H]³⁺=2219.2 [M+4H]⁴⁺=1665.9; [M+5H]⁵⁺=1332.8 (MWcalculated=6660 g/mol)

8f MS: [M+3H]³⁺=2229.7 [M+4H]⁴⁺=1673.3; [M+5H]⁵⁺=1337.7 (MWcalculated=6689 g/mol)

8g MS: [M+3H]³⁺=2218.7 [M+4H]⁴⁺=1664.9 (MW calculated=6659 g/mol)

Synthesis of Compounds 9a to 9g (Mono-Pegylated Insulin Compounds)

70 μl 500 μM 8a in 1/4 (v/v) acetonitrile/water were mixed with 7 μl 10mM maleimide-PEG5k in 1/4 (v/v) acetonitrile/water and 10 μl 0.5 Msodium phosphate buffer pH 7.0 and incubated for 15 min. Compound 9a waspurified by SEC (column: Superdex 200, flow rate: 0.75 ml/min) using 10mM HEPES buffer (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.005% Tween asmobile phase. The collected eluate (approximately 1.5 ml) was directlyused as such for release rate determination.

9b, 9c, 9d, 9e, 9f, or 9g were synthesized as described above from 8b,8c, 8d, 8e, 8f, or 8g, respectively.

9a through 9g: SEC retention time: 19.5 min

Synthesis of Compounds 11a and 11b

11a R1=R2=Me

11b R1=Me, R2=H

3,5-Dimethyl-4-hydroxy-acetophenone (5.0 mmol) (10a) and CuBr₂ (1.7 g,7.5 mmol) were dissolved in 10 ml ethyl acetate and refluxed for 2 h.Solid byproducts were removed by filtration. The filtrate was evaporatedand crude product 11a was purified by RP-HPLC.

11b was synthesized from 4-hydroxy-3-methyl-acetophenone (10b) (0.75 g,5.0 mmol) as described above.

11a: Yield 754 mg (62%)

MS [M+H]⁺=243.1/245.1 (MW+H calculated=244.1 g/mol)

11b: Yield 533 mg (47%)

MS [M+H]⁺=229.2/231.1 (MW+H calculated=230.1 g/mol)

Synthesis of Compounds 12a and 12b

12a R1=R2=Me

12b R1=Me, R2=H

500 mg 11a (2.06 mmol) and 576 mg (4.11 mmol) hexamethylenetetraminewere dissolved in 20 ml of trichloromethane and refluxed for 30 min. Thesolvent was removed in vacuo. 4 ml ethanol and 2 ml of concentrated HClwere added and the slurry was heated to 50° C. for 4 h. The mixture wasconcentrated in vacuo, diluted with acetonitrile/water and 12a waspurified by RP-HPLC.

12b was synthesized from 472 mg (2.06 mmol) 11b as described above.

12a: Yield 547 mg (81%) as TFA-salt

MS [M+Na]⁺=202.2 (MW+Na calculated=202.2 g/mol)

12b: Yield 455 mg (70%) as TFA-salt

MS [M+Na]⁺=188.2 (MW+Na calculated=188.2 g/mol)

Synthesis of Compound 13

500 mg (1.71 mmol) 12a (TFA salt) were dissolved in 10 ml 1/1 (v/v)methanol/water, 129 mg (3.41 mmol) NaBH₄ were added and the mixture wasstirred for 30 min at RT. 0.5 ml of acetic acid were added and 13 waspurified by RP-HPLC.

13: Yield 313 mg (62%) as TFA-salt

MS [M+Na]⁺=204.2 (MW+Na calculated=204.2 g/mol)

NMR (300 MHz, DMSO-d₆) δ[ppm]=8.25 (s, 1H, Phenol), 7.84 (bs, 3H, NH₃⁺), 6.89 (s, 2H, CH_(ar)), 5.85 (d, 1H, Hydroxyl, J=3.7 Hz), 4.62 (m,1H, CH_(Benzyl)), 2.93 (m, 1H, CH_(a)), 2.80 (m, 1H, CH_(b)), 2.17 (s,6H, CH₃).

Synthesis of Compounds 14a to 14d

14a R1=R2=Me, R7=H, R4=Mmt

14b R1=R2=H, R7=Me, R4=Mmt

14c R1=OMe, R2=H, R7=Me, R4=Mmt

14d R1=H, R2=H, R7=Me, R4=Trt

13 (TFA salt, 159 mg, 0.541 mmol) was coupled to compound 2 as describedfor compound 3a to yield 14a.

14b, or 14c were synthesized as described above using synephrine (335mg, 2.00 mmol) or metanephrine (HCl salt, 281 mg, 1.20 mmol),respectively.

Synephrine (335 mg, 2.3 mmol) was coupled to 3-tritylsulfanyl-propionicacid as described above to yield 14d.

14a: Yield 254 mg (87%)

MS [M+Na]⁺=564.7 (MW+Na calculated=564.3 g/mol)

14b: Yield 760 mg (72%)

MS [M+Na]⁺=550.2 (MW+Na calculated=550.3 g/mol)

14c: Yield 530 mg (80%)

MS [M+Na]⁺=580.4 (MW+Na calculated=580.4 g/mol)

14d: Yield 567 mg (49%)

MS [M+Na]⁺=520.5 (MW+Na calculated=520.7 g/mol)

Synthesis of Compounds 15c, 15d and 15f

General Synthesis Protocol:

1 g 2-chlorotrityl chloride resin (loading 1.4 mmol/g) was incubated for1 h with N,N′-dimethylpropane-1,3-diamine (for synthesis of 15c), orN,N′-diethyl-propane-1,3-diamine (for synthesis of 15d) orN,N′-dimethyl-ethane-1,2-diamine (4 eq) (for synthesis of 15f) in DCM.After washing of the resin with DMF, amines were acetylated with 1/1/2(v/v/v) acetic anhydride/pyridine/DMF for 14 h. The resin was washedwith THF and dried. LiAlH₄ (1 M in THF, 4 eq) was added dropwise to thesuspended resin in THF. The resulting suspension was stirred for 3 h at45° C. under nitrogen atmosphere. After cooling, aqueous Rochelle's saltsolution was added and the resin was separated and dried. Compounds werecleaved from resin with 2/1 (v/v) HFIP/DCM (2×30 min). The volatilecomponents were evaporated and the products 15c, 15d or 15f were used inthe following steps without further purification.

15c MS [M+H]⁺=131.2 (MW=130.1 g/mol)

15d MS [M+H]⁺=159.2 (MW=158.1 g/mol)

15f MS [M+H]⁺=117.1 (MW=116 g/mol)

Synthesis of Compounds 16a to 16f and 16i

16a R1=R2=R5=Me, R6=2-(dimethylamino)ethyl, R7=H, R4=Mmt

16b R1=OMe, R2=H, R5=Et, R6=2-(diethylamino)ethyl, R7=H, R4=Mmt

16c R1=OMe, R2=H, R5=Me, R6=3-(N-ethyl-N-methylamino)propyl, R7=Me,R4=Mmt

16d R1=R2=H, R5=Me, R6=3-(N-ethyl-N-methylamino)propyl, R7=Me, R4=Mmt

16e R1=OMe, R2=H, R5=Et, R6=3-(diethylamino)propyl, R7=Me, R4=Mmt

16f R1=R2=H, R5=Et, R6=3-(diethylamino)propyl, R7=Me, R4=Mmt

16i R1=R2=H, R5=Et, R6=2-(diethylamino)ethyl, R7=Me, R4=Trt

14a (120 mg, 0.222 mmol) was dissolved in 1.5 ml of dry THF.p-Nitrophenyl-chloroformate (45 mg, 0.222 mmol) and DIEA (113 μl, 0.665mmol) were added and the mixture was stirred for 30 min at RT. 15a(N,N,N′-trimethyl-ethylene-1,2-diamine) (72 μl, 0.554 mmol) was addedand stirring was continued for 30 min. The solvent was removed in vacuo,100 μl of AcOH were added and 16a was purified by RP-HPLC.

16b was synthesized as described above from 3b (80 mg, 0.15 mmol) and15b (N,N,N′-triethyl-ethylene-1,2-diamine) (55 mg, 0.38 mmol).

16c or 16d were synthesized as describe above from 14c (56 mg, 0.1 mmol)or 14b (53 mg, 0.1 mmol), respectively, and diamine 15c.

16e or 16f were synthesized as described above from 14c (56 mg, 0.1mmol) or 14b, respectively, (53 mg, 0.1 mmol) and diamine 15d.

16i was synthesized as described above from 14d (350 mg, 0.7 mmol) and15b (N,N,N′-triethyl-ethylene-1,2-diamine) (180 μl, 1 mmol).

16a: Yield 120 mg (69%) as TFA salt

MS [M+Na]⁺=692.4 (MW+Na calculated=692.9 g/mol)

16b: Yield 48 mg (40%) as TFA salt

MS [M+Na]⁺=736.3 (MW+Na calculated=736.4 g/mol)

16c: Yield 8 mg (10%) as TFA salt

MS [M+Na]⁺=736.4 (MW+Na calculated=736.4 g/mol)

16d: Yield 20 mg (25%) as TFA salt

MS [M+Na]⁺=706.3 (MW+Na calculated=706.3 g/mol)

16e: Yield 2 mg (3%) as TFA salt

MS [M+Na]⁺=764.6 (MW+Na calculated=764.4 g/mol)

16f: Yield 6 mg (8%) as TFA salt

MS [M+Na]⁺=734.4 (MW+Na calculated=734.3 g/mol)

16i: Yield 152 mg (28%) as TFA salt

MS [M+Na]⁺=690.5 (MW+Na calculated=690.9 g/mol)

Synthesis of Compound 17

Amine 12b (TFA salt) was coupled to compound 2 as described for compound3a.

17: Yield 608 mg (74%)

MS [M+Na]⁺=548.3 (MW+Na calculated=548.7 g/mol)

Synthesis of Compounds 18a and 18b

18a R1=R5=Me, R2=H, R6=3-(dimethylamino)propyl

18b R1=R5=Me, R2=H, R6=2-(N-ethyl-N-methylamino)ethyl

383 mg (0.729 mmol) of 17 were reacted with p-nitrophenyl-chloroformateand N,N,N′-trimethyl-propane-1,3-diamine (15e) or 15f, respectively, toyield 18a or 18b as described for compound 16a.

18a: Yield 287 mg (50%) as TFA salt

MS [M+Na]⁺=690.7 (MW+Na calculated=690.9 g/mol)

18b: Yield 148 mg (26%) as TFA salt

MS [M+Na]⁺=690.9 (MW+Na calculated=690.9 g/mol)

Synthesis of Compounds 16g and 16h

16g R1=R5=Me, R2=H, R6=3-(dimethylamino)propyl

16h R1=R5=Me, R2=H, R6=2-(N-ethyl-N-methylamino)ethyl

18a (287 mg, 0.367 mmol, TFA salt) was dissolved in 5 ml methanol, NaBH₄(41 mg, 1.07 mmol) was added and the mixture was stirred for 30 min atRT. 0.5 ml of acetic acid were added and 16g was purified by RP-HPLC.

18b (8 mg, 0.010 mmol, TFA salt) was reacted as described above to yield16h.

16g: Yield 201 mg (70%) as TFA-salt

MS [M+Na]⁺=692.7 (MW+Na calculated=692.9 g/mol)

16h: Yield 6 mg (77%) as TFA-salt

MS [M+Na]⁺=692.7 (MW+Na calculated=692.9 g/mol)

Synthesis of Compounds 19a to 19i

19a R1=R2=R5=Me, R6=2-(dimethylamino)ethyl, R7=H, R4=Mmt

19b R1=OMe, R2=H, R5=Et, R6=2-(diethylamino)ethyl, R7=H, R4=Mmt

19c R1=OMe, R2=H, R5=Me, R6=3-(N-ethyl-N-methylamino)propyl, R7=Me,R4=Mmt

19d R1=R2=H, R5=Me, R6=3-(N-ethyl-N-methylamino)propyl, R7=Me, R4=Mmt

19e R1=OMe, R2=H, R5=Et, R6=3-(diethylamino)propyl, R7=Me, R4=Mmt

19f R1=R2=H, R5=Et, R6=3-(diethylamino)propyl, R7=Me, R4=Mmt

19g R1=R5=Me, R2=H, R6=3-(dimethylamino)propyl, R7=H, R4=Mmt

19h R1=R5=Me, R2=H, R6=2-(N-ethyl-N-methylamino)ethyl, R7=H, R4=Mmt

19i R1=R2=H, R5=Et, R6=2-(diethylamino)ethyl, R7=Me, R4=Trt

Carbonates 19a to 19i were synthesized from 16a to 16i, respectively, asdescribed for compound 7a.

19a: Yield 98 mg (72%) as TFA-salt

MS [M+Na]⁺=857.8 (MW+Na calculated=858.0 g/mol)

19b: Yield 6 mg (11%) as TFA-salt

MS [M+Na]⁺=901.8 (MW+Na calculated=901.5 g/mol)

19c: Yield 1 mg (15%) as TFA-salt

MS [M+Na]⁺=901.4 (MW+Na calculated=901.5 g/mol)

19d: Yield 8 mg (29%) as TFA-salt

MS [M+Na]⁺=871.4 (MW+Na calculated=871.4 g/mol)

19e: Yield 0.3 mg (18%) as TFA-salt

MS [M+Na]⁺=929.4 (MW+Na calculated=929.5 g/mol)

19f: Yield 4 mg (45%) as TFA-salt

MS [M+Na]⁺=899.7 (MW+Na calculated=899.6 g/mol)

19g: Yield 6 mg (6%) as TFA-salt

MS [M+Na]⁺=857.8 (MW+Na calculated=858.0 g/mol)

19h: Yield 0.8 mg (11%) as TFA-salt

MS [M+Na]⁺=857.7 (MW+Na calculated=858.0 g/mol)

19i: Yield 77 mg (49%) as TFA-salt

MS [M+Na]⁺=856.2 (MW+Na calculated=856.0 g/mol)

Synthesis of Compounds 20a to 20f

20a R1=R2=R5=Me, R6=2-(dimethylamino)ethyl, R7=H

20b R1=OMe, R2=H, R5=Et, R6=2-(diethylamino)ethyl, R7=H

20c R1=OMe, R2=H, R5=Me, R6=3-(N-ethyl-N-methylamino)propyl, R7=Me

20d R1=R2=H, R5=Me, R6=3-(N-ethyl-N-methylamino)propyl, R7=Me

20e R1=R5=Me, R2=H, R6=3-(dimethylamino)propyl, R7=H

20f R1=R5=Me, R2=H, R6=2-(N-ethyl-N-methylamino)ethyl, R7=H

Insulin Derivatives 20a, 20b, 20c, 20d, 20e, or 20f were synthesizedfrom 19a, 19b, 19c, 19d, 19g, or 19h respectively, as described forcompound 8a.

20a MS [M+3H]³⁺=2077.3 [M+4H]⁴⁺=1 559.2 (MW calculated=6231.3 g/mol)

20b MS [M+3H]³⁺=2093.0 [M+4H]⁴⁺=1 569.6 (MW calculated=6274 g/mol)

20c MS [M+3H]³⁺=2090.8 [M+4H]⁴⁺=1 568.7 (MW calculated=6274 g/mol)

20d MS [M+3H]³⁺=2081.3 [M+4H]⁴⁺=1 561.8 (MW calculated=6244 g/mol)

20e MS [M+3H]³⁺=2077.1 [M+4H]⁴⁺=1 558.2 (MW calculated=6231.3 g/mol)

20f MS [M+3H]³⁺=2076.7 [M+4H]⁴⁺=1 559.3 (MW calculated=6231.3 g/mol)

Synthesis of Compounds 21a to 21f (Mono-Pegylated Insulin Derivatives

21a R1=R2=R5=Me, R6=2-(dimethylamino)ethyl, R7=H

21b R1=OMe, R2=H, R5=Et, R6=2-(diethylamino)ethyl, R7=H

21c R1=OMe, R2=H, R5=Me, R6=3-(N-ethyl-N-methylamino)propyl, R7=Me

21d R1=R2=H, R5=Me, R6=3-(N-ethyl-N-methylamino)propyl, R7=Me

21e R1=R5=Me, R2=H, R6=3-(dimethylamino)propyl, R7=H

21f R1=R5=Me, R2=H, R6=2-(N-ethyl-N-methylamino)ethyl, R7=H

Insulin derivatives 21a, 21b, 21c, 21d, 21e, or 21f were synthesizedfrom compound 20a, 20b, 20c, 20d, 20e, or 20f, respectively, asdescribed for compound 9a.

21a through 21f: SEC retention time: 19.5 min

Synthesis of Compounds 23a and 23b

23a R1=Me, R2=H

23b R1=R2=Me

o-Cresol (22a) (1 eq), succinic anhydride (1 eq), and AlCl₃ (3 eq) innitrobenzene were heated to 100° C. for 1 h. The reaction mixture waspoured on HCl/ice and extracted with ether. The organic layer wasextracted with 1 N NaOH and the aqueous layer was acidified withconcentrated HCl. The aqueous layer was extracted with ether and theether was evaporated. 23a was purified by RP-HPLC.

23b was synthesized from 2,6-dimethylphenol (22b) as described above.

23a: Yield 552 mg (31%)

MS [M+Na]⁺=231.0 (MW+Na calculated=231.2 g/mol)

NMR (300 MHz, DMSO-d₆) δ[ppm]=12.05 (bs, 1H, CO₂H), 10.23 (s, 1H, phenolOH), 7.74 (s, 1H, CH_(ar)), 7.7 (d, 1H, CH_(ar), ³J_(H,H)=8.4 Hz), 6.86(d, 1H, CH_(ar), ³J_(H,H)=8.4 Hz), 3.13 (t, 2H, C(O)CH₂, ³J_(H,H)=6.4Hz), 2.53 (t, 2H, CH₂CO₂, ³J_(H,H)=6.4 Hz), 2.16 (s, 3H, CH₃)

23b: Yield 166 mg (15%)

MS [M+Na]⁺=245.4 (MW+Na calculated=245.2 g/mol)

Synthesis of Compound 24

1.85 g (16.02 mmol) cysteamine hydrochloride were dissolved in 15 ml ofTFA and 2.47 g (8.01 mmol) MmtCl were added. After stirring the mixtureat RT for 20 min the solvent was evaporated in vacuo. The residue wasdissolved in diethyl ether and extracted with saturated aqueous NaHCO₃,1N H₂SO₄ and brine. The solvent was evaporated and 24 was purified byRP-HPLC.

24: Yield 1.11 g (30%) as TFA salt

TLC (AcOEt/Et₃N 99/1), R_(f)=0.24

Synthesis of Compounds 25a and 25b

25a R1=Me, R2=H

25b R1=R2=Me

23a (1 eq), HOBt (1.1 eq) and DIC (1 eq) were dissolved in DMF andstirred at RT for 30 min. 24 (TFA salt, 1 eq) and DIEA (3 eq) were addedand the solution was stirred for 60 min. Acetic acid was added and 25awas purified by RP-HPLC.

25b was synthesized from 23b as described above.

25a: Yield 552 mg (25%)

MS [M+Na]⁺=562.7 (MW+Na calculated=564.7 g/mol)

25b: Yield 15 mg (40%)

MS [M+Na]⁺=576.6 (MW+Na calculated=576.6 g/mol)

Synthesis of Compounds 26a and 26b

26a R1=Me, R2=H, R5=R6=3-(dimethylamino)propyl

26b R1=R2=R5=Me, R6=2-(dimethylamino)ethyl

267 mg (0.495 mmol) 25a was reacted with p-nitrophenylchloroformate andN-(3-dimethylamino-propyl)-N′,N′dimethyl-propane-1,3-diamine (15g) toyield 26a as described for compound 16a.

26b was synthesized as described above using 15 mg 25b andN,N,N′-trimethyl-ethane-1,2-diamine (15a).

26a: Yield 282 mg (58%) as double TFA salt

MS [M+Na]⁺=775.2 (MW+Na calculated=776.0 g/mol)

26b: Yield 17 mg (70%) as TFA salt

MS [M+Na]⁺=704.5 (MW+Na calculated=704.6 g/mol)

Synthesis of Compounds 27a and 27b

27a R1=Me, R2=H, R5=R6=3-(dimethylamino)propyl

27b R1=R2=R5=Me, R6=2-(dimethylamino)ethyl

26a (272 mg, 0.277 mmol, double TFA salt) was dissolved in 5 mlmethanol, NaBH₄ (42 mg, 1.09 mmol) was added and the mixture was stirredfor 30 min at RT. 0.5 ml of acetic acid were added and 27a was purifiedby RP-HPLC.

Alcohol 27b was synthesized likewise from 26b (17 mg, 25 μmol, TFAsalt).

27a: Yield 142 mg (52%) as double TFA salt

MS [M+Na]⁺=777.9 (MW+Na calculated=778.0 g/mol)

27b: Yield 6 mg (40%) as TFA salt

MS [M+Na]⁺⁼706.5 (MW+Na calculated=706.6 g/mol)

Synthesis of Compounds 28a and 28b

28a R1=Me, R2=H, R5=R6=3-(dimethylamino)propyl

28b R1=R2=R5=Me, R6=2-(dimethylamino)ethyl

Carbonates 28a or 28b were synthesized from 27a or 27b, respectively, asdescribed for compound 7a.

28a: Yield 1 mg (29%)

MS [M+Na]⁺=942.9 (MW+Na calculated=943.2 g/mol)

28b: Yield 1.5 mg (19%)

MS [M+Na]⁺=871.6 (MW+Na calculated=871.7 g/mol)

Synthesis of Compounds 29a and 29b

29a R1=Me, R2=H, R5=R6=3-(dimethylamino)propyl

29b R1=R2=R5=Me, R6=2-(dimethylamino)ethyl

Insulin derivatives 29a or 29b were synthesized from 28a or 28b,respectively, as described for compound 8a.

29a MS [M+3H]³⁺=2105.8 [M+4H]⁴⁺=1580.2 (MW calculated=6316.4 g/mol)

29b MS [M+3H]³⁺=2081.8 [M+4H]⁴⁺=1562.4 (MW calculated=6244 g/mol)

Synthesis of Mono-Pegylated Insulin Derivatives 30a and 30b

30a R1=Me, R2=H, R5=R6=3-(dimethylamino)propyl

30b R1=R2=R5=Me, R6=2-(dimethylamino)ethyl

Insulin derivatives 30a or 30b were synthesized from 29a or 29b,respectively, as described for compound 9a.

30a and 30b: SEC retention time: 19.5 min

Synthesis of a Polymeric Prodrug According to Formula Ia with anEster-Linked Masking Group and a Dendritic Carrier (9h)

Synthesis of Compound 31

31 was obtained according to the standard solid-phase synthesisprotocol. The amino acids Fmoc-Dpr(Boc)-OH, Fmoc-Dpr(Fmoc)-OH,Fmoc-Dpr(Fmoc)-OH, Fmoc-Ado-OH, and Fmoc-Dpr(Fmoc)-OH were coupled toNovaSyn TG Sieber amide resin. After final fmoc removal the resin wasagitated with 5 eq maleimidopropionic acid and 5 eq DIC in relation toamino groups in DMF for 30 min. 31 was cleaved from resin withTFA/TES/water 95/3/2 (v/v/v). After evaporation of solvent, product 31was purified by RP-HPLC.

MS: [M+H]⁺=2494.6 (MW calculated=2495.4 g/mol)

Synthesis of Compound 32

Compound 32 was obtained according to the standard solid-phase synthesisprotocol. The amino acids Fmoc-Cys(Mmt)-OH, Fmoc-Dpr(Fmoc)-OH,Fmoc-Dpr(Fmoc)-OH, Fmoc-Ado-OH, and Fmoc-Dpr(Fmoc)-OH were coupled toNovaSynTG Sieber amide resin.

After final fmoc removal the resin was agitated with 3 eqBoc-aminoxyacetic acid, 3 eq DIC, and 3 eq HOBt in relation to aminogroups in DMF for 30 min. 32 was cleaved from resin with DCM/TFA/TES97/1/2 (v/v/v). After addition of 0.8 eq pyridine in relation to TFA thesolvent was evaporated and product 32 was purified by RP-HPLC.

MS: [M+H]⁺=2688.2 g/mol (MW calculated=2688.8 g/mol)

Synthesis of Compound 33

Compound 33 was obtained according to the standard solid-phase synthesisprotocol. The amino acids Fmoc-Dpr(ivDde)-OH, Fmoc-Dpr(Fmoc)-OH,Fmoc-Dpr(Fmoc)-OH, Fmoc-Lys(Fmoc)-OH, and Fmoc-Ado-OH were coupled toNovaSyn TG Sieber amide resin.

After final fmoc removal the resin was agitated with 3 eq3,6,9-trioxadecanoic acid, 3 eq PyBOP, and 6 eq DIEA in relation toamino groups in DMF for 60 min. To cleave the ivDde protecting group,the resin was treated three times with 2% hydrazine in DMF. Afterwashing, 3 eq Fmoc-Ser-OH was coupled with 3 eq DIC and 3 eq HOBt for 30min. After final fmoc removal resin was washed and the product wascleaved from resin with DCM/TFA/TES 88/10/2 (v/v/v). Solvent wasevaporated and the residue was oxidized with 10 eq sodium periodate in3/2 (v/v) 0.1 M sodium phosphate pH 7/acetonitrile for 15 min to yield33. Product 33 was purified by RP-HPLC and lyophilized.

MS: [M+H]⁺=3372.1 g/mol (3372.8 g/mol)

Synthesis of Compound 34

6 mg (2.4 μmol) of compound 31 were dissolved in 1 ml 2/1 (v/v)acetonitrile/0.1 M sodium phosphate buffer pH 7 and 65 mg (24.2 μmol) ofcompound 32 were added. The solution was stirred at room temperature for2 h and then the product purified by RP-HPLC and lyophilized (yield: 45mg (78%)).

The lyophilized product (45 mg) was dissolved in 0.5 ml DMF and 10 μlDIEA were added. 5 mg (30 μmol) 3-maleimidopropionic acid and 4.7 μl (30μmol) DIC in 150 μl DMF were added and the reaction mixture was stirredat room temperature for 20 min, the product purified by RP-HPLC andlyophilized.

The lyophilized product was incubated for 10 min in 95/5 (v/v) TFA/waterand then the solvent was removed in a stream of nitrogen. Product 34 waspurified by RP-HPLC and lyophilized (overall yield for all three steps:20 mg (47%)).

MS: 17700-18200 (broad peak) (MW calculated=17749 g/mol)

Synthesis of Compound 9h

1.5 mg (225 nmol) 8g and 5 mg (280 nmol) 34 were mixed, dissolved in 300μl 2/1 (v/v) 0.1 M sodium phosphate buffer pH 7/acetonitrile andincubated for 15 min at room temperature. The product was purified byRP-HPLC and lyophilized. (yield 4 mg, 160 nmol, 70%)

The lyophilized product was dissolved in 200 μl 0.1 M sodium citratebuffer pH 1.5 and 69 mg (20.5 μmol) 33 in 200 μl 2/1 (v/v)acetonitrile/sodium citrate buffer pH 1.5 were added. The mixture wasstirred at room temperature for 24 h and product 9h was purified by sizeexclusion chromatography (column: Superdex 200, buffer: 10 mM HEPES pH7.4, 0.005% Tween-20, 3 mM EDTA, flow rate: 0.75 ml/min)

SEC elution time: 15 min

Synthesis of Compound 37a

250 mg (0.35 mmol) 2-chlorotrityl chloride resin (loading 1.4 mmol/g)was incubated for 1.5 h with 308 mg (4 eq., 1.4 mmol)4,7,10-trioxatridecane-1,13-diamine in 4 ml DCM to yield 35a. The resinwas washed with DCM and dried. 107 mg (0.7 mmol) HOBt, 110 μl (0.7 mmol)DIC, and 150 mg (0.9 mmol) 5-formyl salicylic acid in 3 ml DMF wereadded and the resulting suspension was stirred for 1 h at RT to yield36a. After washing with DCM and THF, the resin was suspended in 6 ml THFand 3 ml (3 mmol) BH₃ THF (1 M in THF, 8.5 eq.) were added dropwise. Thereaction mixture was stirred for 18 h at 45° C. under nitrogenatmosphere. After cooling 4 ml THF, 0.8 ml DIEA and 1.6 ml MeOH wereadded successively. 210 mg (0.84 mmol) I₂ (as a concentrated THFsolution) were added and the suspension was stirred for 1 h. The resinwas repeatedly washed (three times each) with THF, DMF, MeOH, and DCM.The dried resin was reacted with 107 mg (0.7 mmol) HOBt, 110 μl (0.7mmol) DIC, and 55 μL (0.9 mmol) AcOH in 3 ml DMF for 1 h. After washingof the resin with DMF and DCM compound 37a was cleaved from resin with2/1 (v/v) HFIP/DCM (two times for 30 min). The volatile components wereevaporated and the product 37a was used in the following step withoutfurther purification.

37a: Yield 29 mg (20%) as TFA salt

MS [M+Na]⁺=421.4 (MW+Na calculated=421.5 g/mol)

Synthesis of Compound 38

24 mg (0.06 mmol) 37a, 31 mg (0.06 mmol) PyBOP, 32 μl (0.18 mmol) DIEA,and 23 mg (0.06 mmol) 2 in 0.5 ml DMF were reacted for 50 min at roomtemperature. After addition of 50 μl acetic acid product 38 was purifiedby RP-HPLC.

38: Yield 7 mg (15%)

MS [M+Na]⁺=781.3 (MW+Na calculated=781.6 g/mol)

Synthesis of Compound 39

300 mg (0.42 mmol) 2-chlorotrityl chloride resin (loading 1.4 mmol/g)was incubated for 1.5 h with 245 mg (4 eq., 1.7 mmol) 1,8-diaminooctanein 4 ml DCM to yield 35b. The resin was washed with DCM and dried. 107mg (0.7 mmol) HOBT, 110 μl (0.7 mmol) DIC, and 150 mg (0.9 mmol)5-formyl salicylic acid in 3 ml DMF were added and the resultingsuspension was stirred for 1 h at RT to yield 36b. After washing withDCM and THF, the resin was suspended in 6 ml THF and 3 ml (3 mmol) BH₃THF (1 M in THF) were added dropwise. The reaction mixture was stirredfor 18 h at 45° C. under nitrogen atmosphere. After cooling 4 ml THF,0.8 ml DIEA and 1.6 ml MeOH were added successively. 210 mg (0.84 mmol)I₂ (as a concentrated THF solution) were added and the suspension wasstirred for 1 h. The resin was repeatedly washed (three times each) withTHF, DMF, MeOH, and DCM. The dried resin was reacted with 107 mg (0.7mmol) HOBT, 110 μl (0.7 mmol) DIC, and 55 μL (0.9 mmol) AcOH in 3 ml DMFfor 1 h. After washing of the resin with DMF and DCM compound 37b, 78 mg(0.39 mmol) p-nitrophenylchloroformate, and 210 μl (1.2 mmol) DIEA in1/1 (v/v) THF/DCM were reacted for 30 min at RT. The separated resin wassuspended in 1/1 (v/v) THF/DCM and 210 μl (1.2 mmol)N,N,N′-trimethylethylendiamine were added. The resulting suspension wasstirred for 25 min at RT. The resin was separated and washed with DCM.Product 39 was cleaved from resin with 2/1 (v/v) HFIP/DCM (two times for30 min). The volatile components were evaporated and the product 39 waspurified by HPLC.

39: Yield 16 mg (8%) as TFA salt

MS [M+Na]⁺=473.5 (MW+Na calculated=473.3 g/mol)

Synthesis of Compound 40a

38 (7 mg, 9 μmol) was dissolved in 200 μl of dry THF.p-Nitrophenylchloroformate (2.0 mg, 10 μmol) and DIEA (4.4 μl, 25 μmol)were added and the mixture was stirred for 30 min at RT.N,N,N′-Triethylethylenediamine (15b) (18 μl, 0.1 mmol) was added andstirring was continued for 30 min. The solvent was removed in vacuo, 10μl of AcOH were added and 40a was purified by RP-HPLC.

40a: Yield 1 mg (11%) as TFA salt

MS [M+Na]⁺=951.1 (MW+Na calculated=951.8 g/mol)

Synthesis of Compound 40b

15 mg (33 μmol) 39, 18 mg (33 μmol) PyBOP, 23 μl (0.13 mmol) DIEA, and13 mg (35 μmol) 2 in 0.5 ml DMF were reacted for 45 min at roomtemperature. After addition of 50 μl acetic acid product 40 was purifiedby RP-HPLC.

40b: Yield 10 mg (37%) as TFA salt

MS [M+H]⁺=811.5 (MW+Na calculated=810.5 g/mol)

Synthesis of Compound 41a and 41b

Carbonate 41a or 41b was synthesized from 40a or 40b as described forcompound 7a

41a: Yield 0.4 mg as TFA salt

MS [M+Na]⁺=1116.8 (MW+Na calculated=1116.9 g/mol)

41b: Yield 2 mg (16%) as TFA salt

MS [M+H]⁺=976.8 (MW calculated=975.8 g/mol)

Synthesis of Compound 42

Insulin derivative 42 was synthesized from 41b as described for compound8a.

42 MS [M+3H]³⁺=2124.5 [M+4H]⁴⁺=1594.6 (MW calculated=6371 g/mol)

Synthesis of Compound 43

Insulin derivative 43 was synthesized from 42 as described for compound9a.

43: SEC retention time: 18.0 min

Synthesis of Rh-Insulin Loaded PEGA Hydrogel 45

Maleimide derivatization of polyacrylamide based hydrogel (PEGA):

NH₂-PEGA hydrogel beads with 0.4 mmol/g loading and 150-300 μm bead sizewere purchased from Novabiochem.

2.5 g methanol-wet NH₂-PEGA-hydrogel (0.4 mmol/g NH₂-loading) wasweighed into a syringe equipped with a polypropylene frit. Maleimideloading was adjusted by acylation employing a mixture of activatedmaleimidopropionic acid and acetic acid as described in the following.The hydrogel was washed 5 times with DMF and reacted with 13.5 mg (0.08mmol) 3-maleimidopropionic acid, 115.2 μl (1.92 mmol) acetic acid and313 μl (2 mmol) DIC in 4 ml DMF for 30 min. The maleimide derivatizedhydrogel 44 was washed 10 times with DMF and DCM and finally withacetonitrile.

30 mg of maleimide derivatized resin 44 (loading 16 μmol/g) was reactedwith 3 mg of compound 20b (480 nmol, 1.06 eq) in 600 μl 20/80 (v/v)acetonitrile/50 mM phosphate buffer (pH 7.4) for 10 min to giverh-insulin loaded hydrogel 45. The hydrogel 45 was washed 5 times with50/50 (v/v) acetonitrile/water and three times with acetonitrile anddried under vacuum.

Synthesis of Rh-Insulin Carbohydrate-Based Hydrogel 46

NHS-activated “Sepharose 4 Fast Flow” hydrogel beads (chemicallycrosslinked agarose, crosslinker epichlorhydrin) were purchased fromAmersham.

1.5 g ethanol-wet Sepharose hydrogel (150 mg dry hydrogel) was weighedinto a syringe equipped with a polypropylene frit and reacted with 1 M4,7,10-trioxatridecane-1,13-diamine in DMF for 30 min. After 5 washingsteps with DMF, hydrogel was reacted with 8.5 mg (0.05 mmol)3-maleimidopropionic acid, 57 μl (0.95 mmol) acetic acid, 151 mg (1mmol) HOBt and 158 μl (1 mmol) DIC in 4 ml DMF for 30 min to givemaleimide derivatized hydrogel. The hydrogel was washed 10 times withDMF and finally with acetonitrile.

1.5 mg 8c was dissolved in 25/75 (v/v) acetonitrile/50 mM phosphatebuffer pH 7.4 and reacted with 10.8 mg maleimide derivatized hydrogelfor 10 min. The rh-insulin loaded hydrogel 46 was washed five times with50/50 (v/v) acetonitrile/water and three times with acetonitrile anddried under vacuum.

Synthesis Scheme of Fluorescein-Insulin-rHSA (50)

Synthesis of Bismaleimide 47

3-Maleimido-propionic acid (92 mg, 0.54 mmol) in 200 μl DMF was reactedwith DIC (78 μl, 0.50 mmol) at RT for 15 min.4,7,10-Trioxa-tridecan-1,13-diamine (43.5 μl, 0.20 mmol) was added andthe mixture was stirred for 30 min at RT.

After addition of 800 μl 1/4 (v/v) acetic acid/water 47 was purified byRP-HPLC.

47: Yield 23 mg (22%)

MS [M+Na]⁺=545.5 (MW+Na calculated=545.6 g/mol)

Synthesis of rHSA-Maleimide (48)

66.5 μl 3 mM rHSA solution in 145 mM NaCl, 32 mM sodium octanoate,0.0015% Tween-80 was mixed with 66.5 μl 0.5 M phosphate buffer pH 7.0.0.41 mg bismaleimide 47 (0.8 μmol) were added and the mixture wasreacted for 15 min at RT. Compound 48 was purified by SEC (column:Superdex 200, flow rate: 0.75 ml/min) using 10 mM HEPES buffer pH 7.4,150 mM NaCl, 3 mM EDTA, and 0.005% Tween as mobile phase. (Yield: 2.6 ml77.5 μM 48)

SEC retention time: 17.1 min (280 nm)

ESI-MS=66988 (MW calculated=66984 g/mol)

Synthesis of Fluorescein-Insulin-Linker-Maleimide (49)

40 μl of 2.4 mM bismaleimide 47 in 1/1 (v/v) acetonitrile/water (96nmol) were mixed with 40 μl 0.5 M sodium borate buffer pH 5.8. 24 nmol8f in 16.8 μl 1/1 (v/v) acetonitrile/water were added and the mixturewas incubated for 10 min at RT. 5 μl AcOH were added and 49 was purifiedby RP-HPLC.

ESI-MS=7211 (MW calculated=7211 g/mol)

Synthesis of Fluorescein-Insulin-Linker-rHSA 50

a) from 49 and rHSA

b) from 48 and 8f

a)

30 μl of 80 μM 49 in 1/1 (v/v) acetonitrile/water (2.4 nmol) were mixedwith 70 μl 0.25 M sodium phosphate buffer pH 6.4. 8 μl 3 mM rHSA in 145mM NaCl 32 mM sodium octanoate, 0.0015% Tween-80, (24 nmol) was addedand the mixture was incubated at RT for 20 min.

Compound 50 was purified by SEC (column: Superdex 200, flow rate: 0.75ml/min) using 10 mM HEPES buffer pH 7.4, 150 mM NaCl, 3 mM EDTA, and0.005% Tween as mobile phase.

SEC retention time: 17.3 min (500 nm)

ESI-MS=73676 (MW calculated=73673 g/mol)

b)

SEC eluate of rHSA-maleimide 48 (241 μl, 77.5 μM, 18.7 nmol) was mixedwith 20 μl 0.5 M sodium borate buffer pH 5.8. 14 μl 1.41 mM 8f (19.6nmol) in 1/1 (v/v) acetonitrile/water were added and the mixture wasincubated at RT for 10 min. 1.2 μl 48.5 mM 3-maleimido propionic acid(58 nmol) in 1/1 (v/v) acetonitrile/water were added and compound 50 waspurified by SEC (column: Superdex 200, flow rate: 0.75 ml/min) using 10mM HEPES buffer pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% Tween asmobile phase.

SEC retention time: 17.1 min (500 nm)

ESI-MS=73698 (MW calculated=73673 g/mol)

Synthesis Scheme of rHSA-Linker-GLP-1 53a and 53b

Synthesis of 51a

GLP(7-36) (sequence: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-amide) wassynthesized on Rink-amide resin employing fmoc-strategy (SpecialtyPeptide Laboratories, Heidelberg, Germany). N-terminal fmoc-protectinggroup was removed and the resin was washed with DCM and dried. 118 mgresin (0.11 mmol/g, 13.2 μmol) was suspended in a solution of 50 mg 19i(53 μmol) in 750 μl dry DMSO and 22.4 μl DIEA. 2.1 μl pyridine was addedand the mixture was shaken for 48 h at RT. After washing the resin 6times each with DMF and DCM, cleavage of the peptide from resin andremoval of protecting groups was achieved with 96/2/2 (v/v/v)TFA/triethylsilane/water. Volatiles were removed under nitrogen flow and51a was purified by RP-HPLC and lyophilized.

51a: Yield 4.6 mg (9%)

MS: [M+3H]³⁺=1251.0 (MW calculated=3750.3 g/mol)

Synthesis of 51b

Lys28 ivDde side chain protected GLP(7-36) (sequence:HAEGTFTSDVSSYLEGQAAKEFIAWLVK(ivDde)GR-amide) was synthesized onRink-amide resin employing fmoc-strategy (Specialty PeptideLaboratories, Heidelberg, Germany). N-terminal fmoc-protecting group wasremoved and the resin was washed with DCM and dried. 50 mg resin (0.11mmol/g, 5.5 μmol) was suspended in a solution of 25 mg 19i (26 μmol) in400 μl dry DMSO and 11.2 μl DIEA. 1.1 μl pyridine was added and themixture was shaken for 48 h at RT. After washing the resin six timeswith DMF the ivDde protecting group was cleaved by incubating the resin3 times with 5% hydrazine in DMF for 20 min. Fmoc-8-amino,3,6-dioxaoctanoic acid was coupled according to the standard couplingcycle. Fmoc protecting group was removed and carboxy-fluorescein wascoupled by incubating the resin with 8 mg 5-(and-6)-carboxyfluoresceinsuccinimidyl ester and 2 μl DIEA for 60 min. Resin was washed six timeseach with DMF and DCM. Cleavage of the peptide from resin and removal ofprotecting groups was achieved with 96/2/2 (v/v/v)TFA/triethylsilane/water. Volatiles were removed under nitrogen flow.51b was used for the synthesis of 52 without further purification.

MS: [M+3H]⁴⁺=1064.3, [M+2H]³⁺=1418.3 (MW calculated=4254 g/mol)

Synthesis of 52

Raw material 51b was dissolved in 500 μl 1/1 (v/v) acetonitrile/0.25 Msodium phosphate pH 7 and 8 mgN,N′-bis(3-maleimidopropionyl)-2-hydroxy-1,3-propanediamine were added.The solution was stirred at RT for 15 min and 52 was purified by RP-HPLCand lyophilized.

52: Yield: 5.1 mg

MS [M+3H]⁴⁺=1162.8, [M+2H]³⁺=1549.4 (MW calculated=4645 g/mol)

Synthesis of Compound 53a

30 μl 1.57 mM 48 (47 nmol) in 10 mM HEPES buffer pH 7.4, 150 mM NaCl, 3mM EDTA, and 0.005% Tween were mixed with 10 μl 0.5 M sodium phosphatebuffer pH 7.4. A mixture of 2 μl DMSO and 12 μl of 6.06 mM 51a (73 nmol)in water/acetonitrile 9/1 (v/v) was added and the solution was incubatedat RT for 30 min. 53a was purified by SEC (column: Superdex 200, flowrate: 0.75 ml/min) using 10 mM phosphate buffer pH 7.4, 150 mM NaCl, and0.005% Tween as mobile phase.

SEC retention time: 17.7 min (280 nm)

ESI-MS=70745 (MW calculated=70734 g/mol)

Synthesis of 53b

100 μl 3 mM 52 (300 nmol) in 9/1 50 mM sodium phosphate pH7.0/acetonitrile were mixed with 100 μl 3 mM HSA (300 nmol) and thesolution was incubated at RT for 30 min. 53b was purified by SEC(column: Superdex 200, flow rate: 0.75 ml/min) using 10 mM HEPES bufferpH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% Tween as mobile phase.

SEC retention time: 17.7 min (500 nm)

Synthesis of Compounds 54a and 54b

54a R1=Me

54b R1=H

AlCl₃ (1.05 eq) was suspended in DCM and 6-bromohexanoic acid chloride(1 eq) was added. After stirring at RT for 20 min o-cresol (1 eq) wasadded and the mixture was reacted at RT for 25 min. The reaction mixturewas poured into ice water and extracted with ethyl acetate. Theseparated organic phase was dried over Na₂SO₄ and concentrated in vacuo.Product 54a was purified by silica gel column chromatography usingheptane/ethyl acetate (4/1) as mobile phase.

54b was synthesized as described above using 6-bromohexanoic acidchloride and phenol.

54a: Yield 3.7 g (33%)

MS [M+H]⁺=285.1 and 287.2 (MW+H calculated=386.2 g/mol)

54b: Yield 620 mg (15%)

MS [M+H]⁺=271.2 (MW calculated=271.0 g/mol)

Synthesis of Compounds 55a and 55b

55a R1=Me

55b R1=H

DBU (105 μl, 701 μmol) was added to a solution of bromide 54a (105 mg,369 μmol) and tritylthiol (204 mg, 738 μmol) in 50 ml dry DMSO. Thereaction mixture was stirred at RT for 40 min and acidified with 1 NH₂SO₄. The aqueous layer was extracted with ethyl acetate andevaporated. 55a was purified by RP-HPLC.

55b was synthesized according to the same protocol using 54b (180 mg,0.66 mmol).

55a: Yield 173 mg (97%)

MS [M+Na]⁺=503.6 (MW+Na calculated=503.7 g/mol)

55b: Yield 160 mg (85%)

MS [M+Na]⁺=489.5 (MW+Na calculated=489.3 g/mol)

Synthesis of Compounds 56a and 56b

56a R1=Me, R2=R3=3-(dimethylamino)propyl,

56b R1=H, R2=Me, R3=3-(N-ethyl-N-methylamino)propyl

56a was prepared from 55a (9 mg, 19 μmol), p-nitrophenyl-chloroformateand bis(3-dimethylamino-propyl)amine (21 μl, 94 μmol) as described forcompound 16a.

56b was synthesized from 55b (160 mg, 0.34 mmol),p-nitrophenyl-chloroformate and N-ethyl-N,N′dimethyl-1,3-propanediamine(15c) as described for compound 16a.

56a: Yield 12 mg (70%) as TFA salt

MS [M+Na]⁺=716.8 (MW+Na calculated=717.0 g/mol)

56b: Yield 80 mg (32%) as TFA salt

MS [M+Na]⁺=645.6 (MW+Na calculated=645.4 g/mol)

Synthesis of Compounds 57a and 57b

57a R1=Me, R2=R3=3-dimethylamino-propyl,

57b R1=H, R2=Me, R3=3-(N-ethyl-N-methylamino)propyl

57a and 57b were synthesized from 56a (12 mg, 13 μmol, double TFA salt)and 56b (80 mg, 110 μmol, TFA salt), respectively, as described forcompound 16g.

57a: Yield 9 mg (75%) as TFA salt

MS [M+Na]⁺=719.0 (MW+Na calculated=718.7 g/mol)

57b: Yield 60 mg (75%) as TFA salt

MS [M+Na]⁺=647.4 (MW+Na calculated=647.4 g/mol)

Synthesis of Compounds 58a and 58b

58a R1=Me, R2=R3=3-(dimethylamino)propyl,

58b R1=H, R2=Me, R3=3-(N-ethyl-N-methylamino)propyl

57a (1 eq, 8 mg, 9 μmol), 4-nitrophenyl chloroformate (3.5 eq, 6 mg, 30μmol), DIEA (6 eq, 9 μl, 52 μmol), and DMAP (1 eq, 1 mg, 9 μmol) werestirred in 1 ml dry DCM at RT for 45 min under nitrogen atmosphere. Thevolatiles were evaporated and acetic acid was added. The mixture wasdissolved in 1/1 (v/v) acetonitrile/water and the carbonate 58a waspurified by RP-HPLC.

Carbonate 58b was prepared likewise from 57b (135 mg, 0.18 mmol).

58a: Yield 7 mg (70%) as TFA salt

MS [M+Na]⁺=883.8 (MW+Na calculated=884.1 g/mol)

58b: Yield 110 mg (77%) as TFA salt

MS [M+Na]⁺=812.4 (MW+Na calculated=812.5 g/mol)

Synthesis of Compound 59

Rh-Insulin (44.5 mg, 7.7 μmol), carbonate 58a (1 eq, 7 mg, 6.4 mmol),DIEA (15 μl, 88 μmol) and DMAP (1.5 mg, 12 μmol) in 0.3 ml DMSO werereacted at RT for 30 min. The reaction mixture was neutralized withacetic acid and diluted with 1/1 (v/v) acetonitrile/water. RP-HPLCpurification gave the appropriate Trt-protected intermediate.

After lyophilization, the Trt-protected intermediate was mixed with 95/5(v/v) TFA/triethylsilane and stirred for 5 min. Volatiles were removedunder nitrogen flow and 59 was purified by RP-HPLC and lyophilized.Position of insulin modification was verified by DTT reduction and MSanalysis.

59: MS [M+3H]³⁺=2095.5 [M+4H]⁴⁺=1572.2 (MW calculated=6288 g/mol)

Synthesis of Compound 60

60 was prepared from 59 (0.17 μmol) as described for compound 9a.

60: SEC retention time: 19.5 min

Synthesis of Compound 61

57b (70 mg, 90 μmol), DSC (161 mg, 630 μmol), DIEA (192 μl, 1.1 mmol),and DMAP (11 mg, 90 μmol) were stirred in 1 ml dry acetonitrile at RTfor 14 h under nitrogen atmosphere. The volatiles were evaporated andacetic acid was added. The mixture was dissolved in 1/1 (v/v)acetonitrile/water and the carbonate 61 was purified by RP-HPLC.

61: Yield 40 mg (51%) as TFA salt

MS [M+Na]⁺=788.4 (MW+Na calculated=788.5 g/mol)

Synthesis of Compound 62

61 (12 mg, 13 μmol) and NPys-Cl (4 mg, 21 μmol) were stirred in 1 ml DCMat −10° C. for 2 h under nitrogen atmosphere. Volatiles were removedunder nitrogen flow and 62 was purified by RP-HPLC.

62: Yield 7 mg (65%) as TFA salt

MS [M+Na]⁺=700.9 (MW+Na calculated=701.4 g/mol)

Synthesis of Compound 63

0.9 mg desalted rhGH (ProspecTany, Israel, MW 22250 g/mol, 40 nmol) in200 μl mM borate buffer (pH 8.0), 8 μl of carbonate 62 in acetonitrile(38 mM, 300 nmol), and 40 μl DMSO were reacted at RT for 3 h. Thesolvent mixture and low molecular weight compounds were replaced bywater and subsequently by acetate buffer (25 mM, pH 4.2, 0.005% Tween20) by ultrafiltration using Centricon 5 filter (cutoff 5 kDa). 8 μl (80nmol) 10 mM DTT in 25 mM acetate buffer pH 4.2, 0.005% Tween was addedand incubated at RT for 30 min. Low molecular weight compounds wereremoved by ultrafiltration using Centricon 5 filter and 25 rnM acetatebuffer pH 4.2, 0.005% Tween as eluate. After concentration to a volumeof 100 μl(Centricon 5) 20 μl (100 nmol) 5 mM Mal-PEG5k in water and 80μl 0.5 M phosphate buffer pH 7.0 were added. The mixture was incubatedat RT for 5 min. Monoconjugate 63 was separated by SEC (column: Superdex200, flow rate: 0.75 ml/mM) using 10 mM phosphate buffer pH 7.4, 150 mMNaCl, and 0.005% Tween 20 as mobile phase. The collected eluate(approximately 1.0 ml) was diluted with 0.5 ml buffer containing 0.05%NaN₃ and directly used for release rate determination.

63: SEC retention time: 17.5 min

Release of Insulin or Fluorescein-Insulin from Conjugates in Buffer pH7.4

Release of (fluorescein)-insulin from (fluorescein)-insulin conjugates9a to 9h, 21a to 21f, 30a, 30b, 43, 50, and 60, release offluorescein-GLP-1 from 53b, and release of rhGH from 63 was effected bylinker hydrolysis in aqueous buffer pH 7.4. Collected SEC eluates of(fluorescein)-insulin conjugates (see above), fluorescein-GLP-1conjugate and rhGH-conjugate, respectively, were incubated at 37° C. andsamples were taken at time intervals and analyzed by RP-HPLC (insulinconjugates) or SEC (rhGH conjugate, fluorescein insulin conjugates andfluorescein-GLP-1 conjugate) and UV detection at 215 or 280 nm or VISdetection at 500 nm. Peaks correlating with the retention time of nativeinsulin, fluorescein-insulin, fluorescein-GLP-1, and rhGH, respectively,were integrated and plotted against incubation time, and curve-fittingsoftware was applied to estimate the corresponding halftime of release.

Release of Insulin from Hydrogel Conjugates 45 and 46

4 mg of 45 or 2 mg 46 were weighed into a test tube and incubated with 1ml 10 mM HEPES buffer pH 7.4, 150 mM NaCl, 0.005% Tween at 37° C. 45 μlsamples were taken at different time intervals and quantitativelyanalyzed for rh-insulin by a RP-HPLC assay. The rh-insulin peaks wereintegrated and rh-insulin concentration was obtained from a standardcurve. A first order release kinetic was fitted to the data points togive the linker half life.

MS-Analysis of Released Insulin from Compound 9a, 9b and 30a

Samples of buffer released insulin (see above) were analyzed by massspectrometry. FIG. 11 shows the mass spectra of released insulin fromcompound 9a, 9b, and 30a. The mass spectrum of insulin released fromcompound 9a clearly shows a major side product (indicated by arrows),corresponding to the irreversibly pentanoyl-modified insulin. In thiscase, removal of the pentanoyl masking group was not by hydrolysis butby acyl transfer to the insulin. The mass spectrum of insulin releasedfrom compound 9b and 30a shows no modification.

Release of Fluorescein-Insulin from Conjugate 9d and 9e in 80% HumanPlasma

Release of fluorescein-insulin from 9d or 9e was effected by hydrolysisin 80% human plasma in 20 mM HEPES pH 7.4 at 37° C. Samples were takenat time intervals and analyzed by SEC and VIS detection at 500 nm. Peakscorrelating with the retention time of fluorescein-insulin wereintegrated and plotted against incubation time, and curve-fittingsoftware was applied to estimate the corresponding halftime of release.

TABLE Polymeric prodrug hydrolysis compound t_(1/2) buffer pH 7.4t_(1/2) human plasma  9a 40 h nd  9b 55 h nd  9c 4.5 d  nd  9d  7 h  4 h 9e 55 h 30 h  9f 90 h nd  9g 37 h nd  9h 88 h nd 21a 64 d nd 21b  8 dnd 21c 52 d nd 21d 29 d nd 21e 100 d  nd 21f 83 h nd 30a 17 d nd 30b >70d  nd 43  4 h nd 45  7 d nd 46  4 d nd 50 57 h nd 53b 19 h nd 60 10 d nd63 51 d nd nd = not determined

The foregoing is considered illustrative of the principles of theinvention and since numerous modifications will occur to those skilledin the art, it is not intended to limit the invention to the exactconstruction and operation described. All suitable modifications andequivalents fall within the scope of the claims.

Abbreviations:

Boc t-butyloxycarbonyl DBU 1,3-diazabicyclo[5.4.0]undecene DCMdichloromethane (iv)Dde1-(4,4-dimethyl-2,6-dioxo-cyclohexyliden)3-methyl-butyl DICdiisopropylcarbodiimide DIEA diisopropylethylamine DMAPdimethylamino-pyridine DMF N,N-dimethylformamide DMSO dimethylsulfoxideDpr diaminopropionic acid DSC disuccinidylcarbonate EDTAethylenediaminetetraacetic acid Et ethyl eq stoichiometric equivalentfmoc 9-fluorenylmethoxycarbonyl Fmoc-Ado-OHFmoc-8-amino-3,6-dioxaoctanoic acid HFIP hexafluoroisopropanol HEPESN-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) HOBtN-hydroxybenzotriazole LCMS mass spectrometry-coupled liquidchromatography Mal maleimidopropionyl Me methyl Mmt 4-methoxytrityl MSmass spectrum MW molecular mass Npys 3-nitro-2-pyridinesulfenyl PyBOPbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphaterHSA recombinant human serum albumin rhGH recombinant human growthhormone RP-HPLC reversed-phase high performance liquid chromatography RTroom temperature SEC size exclusion chromatography Sucsuccinimidopropionyl TES triethylsilane TFA trifluoroacetic acid THFtetrahydrofurane UV ultraviolet VIS visual Z benzyloxycarbonyl

1. A polymeric cascade prodrug or corresponding linker reagent, whereinthe prodrug or corresponding linker reagent corresponds to a structureselected from the general formula I and II:

wherein X represents a spacer moiety; T represents D or A, wherein Drepresents an amine-containing biologically active moiety and Arepresents a leaving group; Y₁ and Y₂ each independently represent O, Sor NR₆; Y₃ and Y₅ independently represent O or S; Y₄ represents O, NR₆or —C(R₇)(R₈); R2 and R3 represent a moiety selected from the groupconsisting of hydrogen, substituted or unsubstituted linear, branched orcyclical alkyl or heteroalkyl groups, aryls, substituted aryls,substituted or unsubstituted heteroaryls, cyano groups, nitro groups,halogens, carboxy groups, carboxyalkyl groups, alkylcarbonyl groups orcarboxamidoalkyl groups; R₄ represents a moiety selected from the groupconsisting of hydrogen, substituted or unsubstituted linear, branched orcyclical alkyls or heteroalkyls, aryls, substituted aryls, substitutedor unsubstituted heteroaryl, substituted or unsubstituted linear,branched or cyclical alkoxys, substituted or unsubstituted linear,branched or cyclical heteroalkyloxys, aryloxys or heteroaryloxys, cyanogroups and halogens; R₇ and R₈ are each independently selected from thegroup consisting of hydrogen, substituted or unsubstituted linear,branched or cyclical alkyls or heteroalkyls, aryls, substituted aryls,substituted or unsubstituted heteroaryls, carboxyalkyl groups,alkylcarbonyl groups, carboxamidoalkyl groups, cyano groups, andhalogens; R₆ represents a group selected from hydrogen, substituted orunsubstituted linear, branched or cyclical alkyls or heteroalkyls,aryls, substituted aryls and substituted or unsubstituted heteroaryls;R₁ represents a polymer; W represents a group selected from substitutedor unsubstituted linear, branched or cyclical alkyls, aryls, substitutedaryls, substituted or unsubstituted linear, branched or cyclicalheteroalkyls, substituted or unsubstituted heteroaryls; Nu represents anucleophile; n represents zero or a positive imager; and Ar represents amulti-substituted aromatic hydrocarbon or multi-substituted aromaticheterocycle.
 2. The prodrug according to claim 1, wherein T represents Dand the amine-containing biologically active moiety is selected from thegroup consisting of small molecule biologically active agents andbiopolymers.
 3. The prodrug according to claim 1, wherein T represents Dand the amine-containing biologically active moiety comprises abiopolymer selected from the group consisting of proteins, polypeptides,oligonucleotides and peptide nucleic acids.
 4. The prodrug according toclaim 1, wherein the amine-containing biologically active moietycomprises a polypeptide selected from the group consisting of ACTH,adenosine deaminase, agalsidase, albumin, alfa-1 antitrypsin, alfa-1proteinase inhibitor, alteplase, anistreplase, ancrod serine protease,antibodies, antithrombin III, antitrypsins, aprotinin, asparaginases,biphalin, bone-morphogenic proteins, calcitonin, collagenase, DNase,endorphins, enfuvirtide, enkephalins, erythropoietins, factor VIIa,factor VIII, factor Villa, factor IX, fibrinolysin, fusion proteins,follicle-stimulating hormones, granulocyte colony stimulating factor,galactosidase, glucagon, glucagon peptides, glucocerebrosidase,granulocyte macrophage colony stimulating factor,phospholipase-activating protein, gonadotropin chorionic, hemoglobins,hepatitis B vaccines, hirudin, hyaluronidases, idumonidase, immuneglobulins, influenza vaccines, interleukins, IL-1 receptor antagonist,insulins, interferons, keratinocyte growth factor, transforming growthfactors, lactase, leuprolide, levothyroxine, luteinizing hormone, lymevaccine, natriuretic peptide, pancrelipase, papain, parathyroid hormone,PDGF, pepsin, platelet activating factor acetylhydrolase, prolactin,protein C, octreotide, secretin, sermorelin, superoxide dismutase,somatropins, somatostatin, streptokinase, sucrase, tetanus toxinfragment, tilactase, thrombins, thymosin, thyroid stimulating hormone,thyrotropin, tumor necrosis factor, TNF receptor-IgG Fc, tissueplasminogen activator, TSH, urate oxidase, urokinase, vaccines, andplant proteins.
 5. The prodrug according to claim 1, wherein thebiologically active moiety comprises a protein prepared by recombinantDNA technology.
 6. The prodrug according to claim 1, wherein thebiologically active moiety comprises a protein selected from the groupconsisting of antibody fragments, single chain binding proteins,catalytic antibodies and fusion proteins.
 7. The prodrug according toclaim 1, wherein the amine-containing biologically active moietyrepresented by D comprises a protein selected from the group consistingof antibodies, calcitonin, G-CSF, GM-CSF, erythropoietins, hemoglobins,interleukins, insulins, interferons, SOD, somatropin, TNF,TNF-receptor-IgC Fc, and glucagon peptides.
 8. The prodrug according toclaim 2, wherein the amine-containing biologically active drug comprisesa small molecule biologically active agent having at least one primaryor secondary amino group selected from the group consisting of centralnervous system-active agents, anti-infective agents, anti-neoplasticagents, antibacterial agents, anti-fungal agents, analgesic agents,contraceptive agents, anti-inflammatory agents, steroidal agents,vasodilating agents, vasoconstricting agents, and cardiovascular agents.9. The prodrug according to claim 2, wherein the amine-containingbiologically active drug comprises a small molecule biologically activeagent selected from the group consisting of daunorubicin, doxorubicin,idarubicin, mitoxantron, aminoglutethimide, amantadine, diaphenylsulfon,ethambutol,sulfadiazin, sulfamerazin, sulfamethoxazol, sulfalen,clinafloxacin, moxifloxacin, ciprofloxaxin, enoxacin, norfloxacin,neomycin B, sprectinomycin, kanamycin A, meropenem, dopamin, dobutamin,lisinopril, serotonin, acivicin and carbutamid.
 10. The polymericcascade prodrug or corresponding linker reagent according to claim 1,wherein each R4 independently represents a substituent selected from thegroup consisting of hydrogen, methyl, ethyl, ethoxy, methoxy, linearalkyls having three or more carbon atoms, cycloalkyls, branched alkylsand C₁₋₆ heteroalkyls.
 11. The polymeric cascade prodrug orcorresponding linker reagent according to claim 1, wherein R1 representsa polymer selected from the group consisting of polyalkyloxy polymers,dextran, chitosan, hyaluronic acid and derivatives thereof, alginate,xylan, mannan, carrageenan, agarose, cellulose, starch, hydroxyethylstarch, carbohydrate-based polymers, polyvinyl alcohols, polyoxazolines,polyanhydrides, poly(ortho esters), polycarbonates, polyurethanes,polyacrylic acids, polyacrylamides, polyacrylates, polymethacrylates,polyorganophosphazenes, polysiloxanes, polyvinylpyrrolidone,polycyanoacrylates, polyesters, polyiminocarbonates, polyaminoacids,collagen, gelatin, copolymers, grafted copolymers, cross-linkedpolymers, and block copolymers thereof.
 12. The polymeric cascadeprodrug or corresponding linker reagent according to claim 1, wherein R1represents a hydrogel.
 13. The polymeric cascade prodrug orcorresponding linker reagent according to claim 1, wherein R1 representsa branched or hyperbranched polymer.
 14. The polymeric cascade prodrugor corresponding linker reagent according to claim 1, wherein R1represents a dendrimer or dense star polymer.
 15. The polymeric cascadeprodrug or corresponding linker reagent according to claim 1, wherein R1represents a biopolymer.
 16. The polymeric cascade prodrug orcorresponding linker reagent according to claim 1, wherein R1 representsa protein.
 17. The polymeric cascade prodrug or corresponding linkerreagent according to claim 16, wherein the protein is selected from thegroup consisting of albumin, antibodies, fibrin, casein and plasmaproteins.
 18. The polymeric cascade prodrug or corresponding linkerreagent according to claim 1, wherein R1 further includes one or morebiologically active substances bound to the polymer.
 19. The polymericcascade prodrug or corresponding linker reagent according to claim 1,wherein R1 has at least one functional group for linkage to X, andwherein the at least one functional group is selected from the groupconsisting of carboxylic acid and activated derivatives thereof, aminogroups, maleimide, thiol, sulfonic acid and derivatives thereof,carbonate and derivatives thereof, carbamate and derivatives thereof,hydroxyl, aldehyde, ketone, hydrazine, isocyanate, isothiocyanate,phosphoric acids and derivatives thereof, phosphonic acids andderivatives thereof, haloacetyls, alkyl halides, acryloyls, arylatingagents, hydroxylamines, disulfides, vinyl sulfones, vinyl ketones,diazoalkanes, diazoacetyl compounds, epoxide, oxirane, and aziridine.20. The polymeric cascade prodrug or corresponding linker reagentaccording to claim 19, wherein the at least one functional group isselected from the group consisting of thiol, maleimide, amino groups,carboxylic acid and derivatives thereof, carbonate and derivativesthereof, carbamate and derivatives thereof, aldehyde, and haloacetyls.21. The polymeric cascade prodrug or corresponding linker reagentaccording to claim 19, wherein the bond formed between X and the atleast one functional group is selected from the group consisting ofdisulfide, S-succinimido, amide, amino, carboxylic ester, sulphonamide,carbamate, carbonate, oxime, hydrazone, urea, thiourea, phosphate, andphosphonate.
 22. The polymeric cascade prodrug or corresponding linkerreagent according to claim 19, wherein the bond formed between X and theat least one functional group is selected from the group consisting ofS-succinimido, amide, carbamate, and urea.
 23. The polymeric cascadeprodrug linker reagent according to claim 1, wherein A is selected fromthe group consisting of chloride, bromide, fluoride, nitrophenoxy,imidazolyl, N-hydroxysuccinimidyl, N-hydroxybenzotriazolyl,N-hydroxyazobenzotriazolyl, pentafluorphenoxy andN-hyroxysulfosuccinimidyl.
 24. The polymeric cascade prodrug orcorresponding linker reagent according to claim 1, wherein

represents a moiety selected from the group consisting of


25. The polymeric cascade prodrug or corresponding linker reagentaccording to claim 1, wherein

represents a moiety selected from the group consisting of


26. The polymeric cascade prodrug or corresponding linker reagentaccording to claim 1, wherein R6 represents an additional Nu-W.
 27. Thepolymeric cascade prodrug or corresponding linker reagent according toclaim 1, wherein

represents a structure selected from the group consisting of

wherein R9, R10, R11 and R12 each independently represent a moietyselected from the group consisting of hydrogen, substituted or non-substituted alkyls or heteroalkyls, and substituted or non-substitutedaryls or heteroaryls, and m represents an integer of 2 to
 10. 28. Thepolymeric cascade prodrug or corresponding linker reagent according toclaim 27, wherein R9, 10, R11 and R12 each independently represent amoiety selected from the group consisting of hydrogen and substituted ornon-substituted alkyls.
 29. The polymeric cascade prodrug orcorresponding linker reagent according to claim 1, wherein Nu representsa nucleophile selected from the group consisting of primary, secondaryand tertiary amino groups, thiols, carboxylic acids, hydroxylamines,hydrazine and nitrogen containing heteroaryls.
 30. The polymeric cascadeprodrug or corresponding linker reagent according to claim 1, wherein Y₄represents —C(R₇)(R₈) and at least one of R7 and R8 is not hydrogen. 31.The polymeric cascade prodrug or corresponding linker reagent accordingto claim 1, wherein Ar represents a structure selected from the groupconsisting of:

wherein each W independently represents O, S, or N.
 32. The polymericcascade prodrug or corresponding linker reagent according to claim 1,wherein Ar represents a monocyclic or dicyclic aromatic hydrocarbon oraromatic heterocycle.
 33. The polymeric cascade prodrug or correspondinglinker reagent according to claim 1, wherein the Ar represents afive-membered or six-membered aromatic hydrocarbon or aromaticheterocycle.
 34. A method for synthesizing a polymeric prodrug, themethod comprising: (a) providing a starting molecule corresponding tothe general Formula II or IIb:

(b) reacting the starting molecule with a masking group having anucleophile to form at least one intermediate compound wherein themasking group is bound to Y₂; and (c) reacting an amine-containingbiologically active moiety D with the at least one intermediate compoundto form a polymeric prodrug; wherein Y₂ is selected from O, S, or NR6;Y₃ is selected from O or S; X is a spacer moiety; R2 and R3 are selectedindependently from hydrogen, substituted or non-substituted linear,branched or cyclical alkyl or heteroalkyl, aryl, substituted ornon-substituted heteroaryl, cyano nitro, halogen, carboxy, carboxyalkyl,alkylcarbonyl or carboxamidoalkyl; R4 is selected from hydrogen,substituted or non-substituted linear, branched or cyclical alkyl orheteroalkyl, aryl, substituted aryl, substituted or non-substitutedheteroaryl, substituted or non-substituted linear, branched, or cyclicalalkoxy, substituted or non-substituted linear, branched, or cyclicalheteroalkyloxy, aryloxy or heteroaryloxy, cyano, or halogen; R6 isselected from hydrogen, substituted or non-substituted linear, branchedor cyclical alkyl or heteroalkyl, aryl, substituted aryl substituted ornon-substituted heteroaryl; n is zero or a positive integer and Ar is amulti-substituted aromatic hydrocarbon or a multi-substituted aromaticheterocycle.
 35. A method for hydrolyzing a polymeric cascade prodrug orcorresponding linker reagent according to claim 1, comprising providingthe prodrug or corresponding linker reagent and placing the prodrug orcorresponding linker reagent in a solution with a pH of approximately7.4.
 36. A method of administering an amine-containing moiety to anorganism in need thereof, the method comprising providing a polymericcascade prodrug according to claim 1, administering the polymericcascade prodrug to the organism and cleaving the amine-containing moietyfrom the polymeric cascade prodrug by means of a substantiallynon-enzymatic reaction.